Methods of preventing and removing trisulfide bonds

ABSTRACT

The present invention pertains to methods of preventing and eliminating trisulfide bonds in proteins such as antibodies. In one embodiment, trisulfide bonds in proteins are converted to disulfide bonds as part of chromatographic purification procedures. In another embodiment, the formation of trisulfide bonds in proteins is inhibited by implementation of methods described herein during the cell culture production of such proteins. In another embodiment, monoclonal antibodies are produced by the methods described herein.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 15/425,056,filed Feb. 6, 2017 and entitled “METHODS OF PREVENTING AND REMOVINGTRISULFIDE BONDS” which is a divisional of application Ser. No.14/117,554, filed Apr. 3, 2014 and entitled “METHODS OF PREVENTING ANDREMOVING TRISULFIDE BONDS” which is a national stage filing under 35U.S.C. § 371 of PCT International Application PCT/US2012/037610, filedMay 11, 2012 and entitled “METHODS OF PREVENTING AND REMOVING TRISULFIDEBONDS,” which claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application No. 61/617,529, filed Mar. 29, 2012 and U.S.provisional application No. 61/485,973, filed May 13, 2011, the contentsof each of which are incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to methods of preventing and eliminatingthe formation of trisulfide bonds in proteins during protein production.

Background Art

Recombinant proteins, and in particular, monoclonal antibodies (mAbs),have become an important class of therapeutic compounds employed for thetreatment of a broad range of diseases. Recent successes in the field ofbiotechnology have improved the capacity to produce large amounts ofsuch proteins. However, extensive characterization of the productsdemonstrates that the proteins are subject to considerableheterogeneity. For example, molecular heterogeneity can result fromchemically-induced modifications such as oxidation, deamidation, andglycation as well post-translational modifications such as proteolyticmaturation, protein folding, glycosylation, phosphorylation, anddisulfide bond formation. Molecular heterogeneity is undesirable becausetherapeutic products must be extensively characterized by an array ofsophisticated analytical techniques and meet acceptable standards thatensure product quality and consistency.

Antibodies (or immunoglobulins) are particularly subject to suchstructural heterogeneity due to the fact that they are large,multi-chain molecules. For example, IgG antibodies are composed of fourpolypeptide chains: two light chain polypeptides (L) and two heavy chainpolypeptides (H). The four chains are typically joined in a “Y”configuration by disulfide bonds that form between cysteine residuespresent in the heavy and light chains. These disulfide linkages governthe overall structure of the native H₂L₂ tetramer. Overall, IgG1antibodies contain four interchain disulfide bonds, including two hingeregion disulfides that link the H chains, and one disulfide bond betweeneach heavy H and L chain. In addition, twelve intrachain disulfidelinkages may involve each remaining cysteine residue present in themolecule. Incomplete disulfide bond formation, or bond breakage viaoxidation or beta-elimination followed by disulfide scrambling, are allpotential sources of antibody heterogeneity. In addition, a further typeof modification, namely trisulfide (—CH₂—S—S—S—CH₂—) bond formation, wasrecently reported within the interchain, hinge region bonds of a humanIgG2 antibody. See, Pristatsky et al., Anal. Chem. 81: 6148 (2009).

Trisulfide linkages have previously been detected in superoxidedismutase (Okado-Matsumoto et al., Free Radical Bio. Med. 41: 1837(2006)), a truncated form of interleukin-6 (Breton et al., J. Chromatog.709: 135 (1995)), and bacterially expressed human growth hormone (hGH)(Canova-Davis et al., Anal. Chem. 68: 4044 (1996)). In the case of hGH,it was speculated that trisulfide formation was promoted by H₂S releasedduring the fermentation process. See, International Published PatentApplication No. WO 96/02570. Consistent with this hypothesis, thetrisulfide content of hGH was increased by exposure to H₂S in solution.See, U.S. Pat. No. 7,232,894. In addition, exposing bioreactor materialor bacterial lysate to an inert gas inhibited trisulfide formation,apparently by stripping H₂S from the system. See, InternationalPublished Patent Application No. WO 2006/069940.

Conditions that influence the level of trisulfide in hGH generatedduring purification from bacteria have previously been reported. Forexample, the presence of alkali metal salts has been reported to inhibitincreases in trisulfide bonds during downstream protein processing. See,U.S. Pat. No. 7,232,894. Furthermore, treatment of hGH withreduction-oxidation (REDOX) compounds, including L-cysteine, in solutionwas found to convert trisulfide bonds to disulfide bonds. See,International Published Patent Application No. WO 94/24157. For example,treatment of purified IgG2 with a 20-fold molar ratio of L-cysteine insolution was found to convert hinge region trisulfide bonds todisulfides during sample preparation for analytical studies. See,Pristatsky et al., Anal. Chem. 81: 6148 (2009).

Unfortunately, removal of trisulfide bonds by exposure to cysteine insolution has several drawbacks, in particular for large scaleprocessing. For example, large quantities of cysteine are required. Thismethod also necessitates a separate step to remove cysteine from thesample after trisulfide bonds are removed. In addition, removal oftrisulfide bonds by exposure to cysteine in solution can promoteaggregation through the formation of undesirable disulfide linkages.Therefore, in order to address the limitations of previous methods forreducing trisulfide bonds, the methods described herein provideefficient and improved means for preventing and eliminating theformation of trisulfide bonds in proteins (such as, for example, inantibodies) during production and purification procedures used in themanufacture of such proteins.

BRIEF SUMMARY OF THE INVENTION

The present invention pertains to methods of preventing and eliminatingtrisulfide bonds in proteins such as antibodies. In one embodiment,trisulfide bonds in proteins are converted to disulfide bonds as part ofchromatographic purification procedures. In another embodiment, theformation of trisulfide bonds in proteins is inhibited by implementationof methods described herein during the cell culture production of suchproteins.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1: Indirect assessment of IgG1 mAb trisulfide content by heatinduced fragmentation. Samples were heated and analyzed by non-reducingcapillary electrophoresis (CE-SDS assay). In each electropherogram, themain peak corresponds to intact H₂L₂ tetramer (indicated by the upperschematic and arrow), and the adjacent peak, which represents the singlemost abundant impurity, has a migration pattern consistent with aspecies lacking one light chain (HHL species; indicated by the lowerschematic and arrow). The percent of total peak area is indicated forthe intact H₂L₂ tetramer and the HHL species, respectively. (A) IgG1mAb-A containing 13.1% H-L trisulfide (percent trisulfide determined bypeptide mapping analysis). (B) IgG1 mAb-A-LT—an alternative preparationof mAb-A in which trisulfide was undetectable by peptide mapping.

FIG. 2: Bar graph displaying the effect of hydrogen sulfide (mM) ontrisulfide levels. Samples were taken from cultures exposed to variableconcentrations of hydrogen sulfide (mM) over various times. Samples werepurified using Protein A and subjected to peptide mapping analysis todetermine the percentage of trisulfide bonds. The bar height representsthe average percentage of antibodies having at least one trisulfide bondbetween the heavy and light chains.

FIG. 3: Bar graph displaying the effect of cysteine concentration ontrisulfide levels. Samples were taken from cell cultures fed with orwithout supplemental cysteine (i.e., complete feed+total amino acidsupplement+supplemental Cys; complete feed+supplemental Cys & Met;complete feed+supplemental Met only). Samples were purified usingProtein A and subjected to peptide mapping analysis to determine thepercentage of trisulfide bonds present. The bar height represents theaverage percentage of antibodies containing at least one trisulfide bondbetween the heavy and light chains.

FIG. 4: Schematic drawings of the disulfide connectivity in human IgGantibodies. (A) IgG1, (B) IgG1, (C) IgG3, and (D) IgG4. Cys residues arenumbered based on their position in the sequence, where “1” is the Cysresidue closest to the N-terminus.

FIG. 5: Extracted ion chromatograms (EIC) and UV trace showing thedisulfide- and trisulfide-linked Lys-C peptide clusters connecting thefifth Cys residues of the light and heavy chains of mAb1. (A-I) the EICof the trisulfide-linked PepLL12/PepHL13; (A-II) the EIC of thedisulfide-linked PepLL12/PepHL13; (A-III) UV profile at 214 nm. Thenumbers shown in each panel are the integrated peak areas. (B) Plot ofthe detected amounts of the trisulfide at the LC5-HC5 linkage in mAb2samples that had been spiked with varying amounts of a mAb2-D containing7.8% trisulfide in the LC5-HC5 linkage. (C) Plot showing reproducibilityof the focused Lys-C peptide mapping method.

FIG. 6: Trisulfides are Stable in vitro in Rat Serum, but RapidlyConvert to Disulfides in vivo.

FIG. 7: Comparison of trisulfide formation in spiked antibodypreparations. Purified antibody which contains 1.8% trisulfide contentwas spiked into cultures in the presence or absence of cells. Trisulfideformation was highest in cultures which did not contain cells(approximately 11%). Addition of cells to the culture decreasedtrisulfide formation.

FIG. 8: Effects of different media additives on release of hydrogensulfide. Pyruvate, methyl pyruvate, ethyl pyruvate, DL-glyceraldehyde,and glyoxylic acid decrease hydrogen sulfide.

FIG. 9: Effect of pyruvate on hydrogen sulfide in solution with cysteineand glucose or cysteine and amino acids. aa #1, aa #2, and aa #3represent subgroupings of amino acids present in the feed liquid.

FIG. 10: Effect of pyruvate on hydrogen sulfide in various media. Allcysteine concentrations were normalized to that in feed liquid. Iscove'sModified Dulbecco's Medium (IMDM) and Dulbecco's Modified Eagle Medium(DMEM) are common cell-culture media.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an antibody” or “a protein” is understoodto represent one or more antibody or protein molecules, respectively. Assuch, the terms “a” (or “an”), “one or more,” and “at least one” can beused interchangeably herein.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Likewise, the term “protein” refers toany chain or chains of two or more polypeptide. Hence, an antibody maybe referred to as “a protein” even though it is comprised of multiplepolypeptides (for example, an IgG antibody is comprised of two heavychain and two light chain polypeptides). Thus, peptides, dipeptides,tripeptides, oligopeptides, “amino acid chain,” or any other term usedto refer to a chain or chains of two or more amino acids, are includedwithin the definition of “protein” or “polypeptide,” and the terms“protein” or “polypeptide” may be used instead of, or interchangeablywith any of these terms. Hence, because the terms “protein” and“polypeptide” encompass plural polypeptides, these terms encompasspolypeptide multimers such as antibodies.

The term “polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation (e.g. modification with N-acetyl glucosamine,galactose, or sialic acid moities), pegylation (i.e. covalent attachmentof polyethylene glycol) acetylation, phosphorylation, amidation,glycation (e.g. dextran modification), derivatization by knownprotecting/blocking groups, proteolytic cleavage, or modification bynon-naturally occurring amino acids. A polypeptide may be derived from anatural biological source or produced by recombinant technology, but isnot necessarily translated from a designated nucleic acid sequence. Itcan be generated in any manner, including by chemical synthesis.

Polypeptides can have a defined three-dimensional structure, althoughthey do not necessarily have such structure. Polypeptides with a definedthree-dimensional structure are referred to as folded, and polypeptideswhich do not possess a defined three-dimensional structure, but rathercan adopt a large number of different conformations, and are referred toas unfolded. As used herein, the term glycoprotein refers to a proteincoupled to at least one carbohydrate moiety that is attached to theprotein via an oxygen-containing or a nitrogen-containing side chain ofan amino acid residue, e.g., a serine residue or an asparagine residue.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for purposed of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

Also included as polypeptides of the present invention are fragments,derivatives, analogs, or variants of the foregoing polypeptides, and anycombination thereof. The terms “fragment,” “variant,” “derivative” and“analog” when referring to antibodies or antibody polypeptides of thepresent invention include any polypeptides which retain at least some ofthe antigen-binding properties of the corresponding native antibody orpolypeptide. Fragments of polypeptides of the present invention includeproteolytic fragments, as well as deletion fragments, in addition tospecific antibody fragments discussed elsewhere herein. Variants ofantibodies and antibody polypeptides of the present invention includefragments as described above, and also polypeptides with altered aminoacid sequences due to amino acid substitutions, deletions, orinsertions. Variants may occur naturally or be non-naturally occurringNon-naturally occurring variants may be produced using art-knownmutagenesis techniques. Variant polypeptides may comprise conservativeor non-conservative amino acid substitutions, deletions or additions.Derivatives of antibodies and antibody polypeptides of the presentinvention, are polypeptides which have been altered so as to exhibitadditional features not found on the native polypeptide. Examplesinclude fusion proteins. Variant polypeptides may also be referred toherein as “polypeptide analogs.” As used herein a “derivative” of anantibody or antibody polypeptide refers to a subject polypeptide havingone or more residues chemically derivatized by reaction of a functionalside group. Also included as “derivatives” are those peptides whichcontain one or more naturally occurring amino acid derivatives of thetwenty standard amino acids. For example, 4-hydroxyproline can besubstituted for proline; 5-hydroxylysine can be substituted for lysine;3-methylhistidine can be substituted for histidine; homoserine can besubstituted for serine; and ornithine can be substituted for lysine.

Proteins secreted by mammalian cells can have a signal peptide orsecretory leader sequence which is cleaved from the mature protein onceexport of the growing protein chain across the rough endoplasmicreticulum has been initiated. Polypeptides secreted by vertebrate cellscan have a signal peptide fused to the N-terminus of the polypeptide,which is cleaved from the complete or “full length” polypeptide toproduce a secreted or “mature” form of the polypeptide. In certainembodiments, the native signal peptide, e.g., an immunoglobulin heavychain or light chain signal peptide is used, or a functional derivativeof that sequence that retains the ability to direct the secretion of thepolypeptide that is operably associated with it. Alternatively, aheterologous mammalian signal peptide, or a functional derivativethereof, may be used. For example, the wild-type leader sequence may besubstituted with the leader sequence of human tissue plasminogenactivator (TPA) or mouse ß-glucuronidase.

The present methods include the use of antibodies, or antigen-bindingfragments, variants, or derivatives thereof. Unless specificallyreferring to full-sized antibodies such as naturally-occurringantibodies, the term “antibodies” encompasses full-sized antibodies aswell as antigen-binding fragments, variants, analogs, or derivatives ofsuch antibodies, e.g., naturally occurring antibody or immunoglobulinmolecules or engineered antibody molecules or fragments that bindantigen in a manner similar to antibody molecules.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. An antibody or immunoglobulin comprises at least the variabledomain of a heavy chain, and normally comprises at least the variabledomains of a heavy chain and a light chain. Basic immunoglobulinstructures in vertebrate systems are relatively well understood. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988).

The term “immunoglobulin” comprises various broad classes ofpolypeptides that can be distinguished biochemically. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, (γ, μ, α, δ, ε) withsome subclasses among them (e.g., γ1-γ4). It is the nature of this chainthat determines the “class” of the antibody as IgG, IgM, IgA IgG, orIgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgG1,IgG2, IgG3, IgG4, IgA1, etc. are well characterized and are known toconfer functional specialization. Modified versions of each of theseclasses and isotypes are readily discernable to the skilled artisan inview of the instant disclosure and, accordingly, are within the scope ofthe instant invention. All immunoglobulin classes are clearly within thescope described herein although the following discussion will generallybe directed to the IgG class of immunoglobulin molecules. With regard toIgG, a standard immunoglobulin molecule comprises two identical lightchain polypeptides of molecular weight approximately 23,000 Daltons, andtwo identical heavy chain polypeptides of molecular weight53,000-70,000. The four chains are typically joined by disulfide bondsin a “Y” configuration wherein the light chains bracket the heavy chainsstarting at the mouth of the “Y” and continuing through the variableregion.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VL) and heavy (VH) chain portions determineantigen recognition and specificity. Conversely, the constant domains ofthe light chain (CL) and the heavy chain (CH1, CH2 or CH3) conferimportant biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen binding site or amino-terminusof the antibody. The N-terminal portion is a variable region and at theC-terminal portion is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the VL domain and VH domain, or subset of the complementaritydetermining regions (CDRs), of an antibody combine to form the variableregion that defines a three dimensional antigen binding site. Thisquaternary antibody structure forms the antigen binding site present atthe end of each arm of the Y. More specifically, the antigen bindingsite is defined by three CDRs on each of the VH and VL chains. In someinstances, e.g., certain immunoglobulin molecules derived from camelidspecies or engineered based on camelid immunoglobulins, a completeimmunoglobulin molecule may consist of heavy chains only, with no lightchains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993).

In naturally occurring antibodies, the six “complementarity determiningregions” or “CDRs” present in each antigen binding domain are short,non-contiguous sequences of amino acids that are specifically positionedto form the antigen binding domain as the antibody assumes its threedimensional configuration in an aqueous environment. The remainder ofthe amino acids in the antigen binding domains, referred to as“framework” regions, show less inter-molecular variability. Theframework regions largely adopt a β-sheet conformation and the CDRs formloops which connect, and in some cases form part of, the β-sheetstructure. Thus, framework regions act to form a scaffold that providesfor positioning the CDRs in correct orientation by inter-chain,non-covalent interactions. The antigen binding domain formed by thepositioned CDRs defines a surface complementary to the epitope on theimmunoreactive antigen. This complementary surface promotes thenon-covalent binding of the antibody to its cognate epitope. The aminoacids comprising the CDRs and the framework regions, respectively, canbe readily identified for any given heavy or light chain variable regionby one of ordinary skill in the art, since they have been preciselydefined (see, “Sequences of Proteins of Immunological Interest,” Kabat,E., et al., U.S. Department of Health and Human Services, (1983); andChothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which areincorporated herein by reference in their entireties).

Antibodies or antigen-binding fragments, variants, or derivativesthereof include, but are not limited to, polyclonal, monoclonal,multispecific, human, humanized, primatized, or chimeric antibodies,single chain antibodies, epitope-binding fragments, e.g., Fab, Fab′ andF(ab′)₂, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (sdFv), fragments comprising either a VL or VHdomain, fragments produced by a Fab expression library, andanti-idiotypic (anti-Id) antibodies. ScFv molecules are known in the artand are described, e.g., in U.S. Pat. No. 5,892,019. Immunoglobulin orantibody molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA,and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) orsubclass of immunoglobulin molecule.

Antibody fragments, including single-chain antibodies, may comprise thevariable region(s) alone or in combination with the entirety or aportion of the following: hinge region, CH1, CH2, and CH3 domains. Alsoincluded are antigen-binding fragments comprising any combination ofvariable region(s) with a hinge region, CH1, CH2, and CH3 domains.Antibodies or immunospecific fragments thereof can be from any animalorigin including birds and mammals. The antibodies can be human, murine,donkey, rabbit, goat, guinea pig, camel, llama, horse, or chickenantibodies. In another embodiment, the variable region may becondricthoid in origin (e.g., from sharks). As used herein, “human”antibodies include antibodies having the amino acid sequence of a humanimmunoglobulin and include antibodies isolated from human immunoglobulinlibraries or from animals transgenic for one or more humanimmunoglobulins and that do not express endogenous immunoglobulins, asdescribed infra and, for example in, U.S. Pat. No. 5,939,598 byKucherlapati et al. A human antibody is still “human” even if amino acidsubstitutions are made in the antibody.

As used herein, the term “heavy chain portion” includes amino acidsequences derived from an immuno globulin heavy chain. A polypeptidecomprising a heavy chain portion comprises at least one of: a CH1domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain,a CH2 domain, a CH3 domain, or a variant or fragment thereof. Forexample, a binding polypeptide for use in the invention may comprise apolypeptide chain comprising a CH1 domain; a polypeptide chaincomprising a CH1 domain, at least a portion of a hinge domain, and a CH2domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; apolypeptide chain comprising a CH1 domain, at least a portion of a hingedomain, and a CH3 domain, or a polypeptide chain comprising a CH1domain, at least a portion of a hinge domain, a CH2 domain, and a CH3domain. In another embodiment, a polypeptide of the invention comprisesa polypeptide chain comprising a CH3 domain. Further, a bindingpolypeptide for use in the invention may lack at least a portion of aCH2 domain (e.g., all or part of a CH2 domain). As set forth above, itwill be understood by one of ordinary skill in the art that thesedomains (e.g., the heavy chain portions) may be modified such that theyvary in amino acid sequence from the naturally occurring immunoglobulinmolecule.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof disclosed herein, the heavy chain portions of onepolypeptide chain of a multimer are identical to those on a secondpolypeptide chain of the multimer. Alternatively, heavy chainportion-containing monomers of the invention are not identical. Forexample, each monomer may comprise a different target binding site,forming, for example, a bispecific antibody.

The heavy chain portions of a binding polypeptide for use in thediagnostic and treatment methods disclosed herein may be derived fromdifferent immunoglobulin molecules. For example, a heavy chain portionof a polypeptide may comprise a CH1 domain derived from an IgG1 moleculeand a hinge region derived from an IgG3 molecule. In another example, aheavy chain portion can comprise a hinge region derived, in part, froman IgG1 molecule and, in part, from an IgG3 molecule. In anotherexample, a heavy chain portion can comprise a chimeric hinge derived, inpart, from an IgG1 molecule and, in part, from an IgG4 molecule.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain. The light chainportion can comprise at least one of a VL or CL domain.

Antibodies, or antigen-binding fragments, variants, or derivativesthereof disclosed herein may be described or specified in terms of theepitope(s) or portion(s) of an antigen, e.g., a target polypeptide thatthey recognize or specifically bind. The portion of a target polypeptidewhich specifically interacts with the antigen binding domain of anantibody is an “epitope,” or an “antigenic determinant.” A targetpolypeptide can comprise a single epitope, at least two epitopes, or anynumber of epitopes, depending on the size, conformation, and type ofantigen. Furthermore, it should be noted that an “epitope” on a targetpolypeptide may be or include non-polypeptide elements, e.g., an“epitope may include a carbohydrate side chain.

Antibodies or antigen-binding fragments, variants or derivatives thereofmay be “multispecific,” e.g., bispecific, trispecific or of greatermultispecificity, meaning that it recognizes and binds to two or moredifferent epitopes present on one or more different antigens (e.g.,proteins) at the same time. Thus, whether an antibody is “monospecific”or “multispecific,” e.g., “bispecific,” refers to the number ofdifferent epitopes with which a binding polypeptide reacts.Multispecific antibodies may be specific for different epitopes of atarget polypeptide described herein or may be specific for a targetpolypeptide as well as for a heterologous epitope, such as aheterologous polypeptide or solid support material.

As used herein the term “valency” refers to the number of potentialbinding domains, e.g., antigen binding domains, present in an antibody.Each binding domain specifically binds one epitope. When an antibodycomprises more than one binding domain, each binding domain mayspecifically bind the same epitope, for an antibody with two bindingdomains, termed “bivalent monospecific,” or to different epitopes, foran antibody with two binding domains, termed “bivalent bispecific.” Anantibody may also be bispecific and bivalent for each specificity(termed “bispecific tetravalent antibodies”). In another embodiment,tetravalent minibodies or domain deleted antibodies can be made.

Bispecific bivalent antibodies, and methods of making them, aredescribed, for instance in U.S. Pat. Nos. 5,731,168; 5,807,706;5,821,333; and U.S. Appl. Publ. Nos. 2003/020734 and 2002/0155537, thedisclosures of all of which are incorporated by reference herein.Bispecific tetravalent antibodies, and methods of making them aredescribed, for instance, in WO 02/096948 and WO 00/44788, thedisclosures of both of which are incorporated by reference herein. Seegenerally, PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO92/05793; Tutt et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos.4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al.,J. Immunol. 148:1547-1553 (1992).

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup. In most naturally occurring IgG molecules, the CH1 and CL regionsare linked by a disulfide bond and the two heavy chains are linked bytwo disulfide bonds at positions corresponding to 239 and 242 using theKabat numbering system (position 226 or 229, EU numbering system).

The term “trisulfide bond” is described further below.

As used herein, the term “chimeric antibody” will be held to mean anyantibody wherein the immunoreactive region or site is obtained orderived from a first species and the constant region (which may beintact, partial or modified in accordance with the instant invention) isobtained from a second species. In some embodiments, the target bindingregion or site will be from a non-human source (e.g. mouse or primate)and the constant region is human.

As used herein, the term “engineered antibody” refers to an antibody inwhich the variable domain in either the heavy and light chain or both isaltered by at least partial replacement of one or more CDRs from anantibody of known specificity and, if necessary, by partial frameworkregion replacement and sequence changing. Although the CDRs may bederived from an antibody of the same class or even subclass as theantibody from which the framework regions are derived, it is envisagedthat the CDRs will be derived from an antibody of different class andpreferably from an antibody from a different species. An engineeredantibody in which one or more “donor” CDRs from a non-human antibody ofknown specificity is grafted into a human heavy or light chain frameworkregion is referred to herein as a “humanized antibody.” It may not benecessary to replace all of the CDRs with the complete CDRs from thedonor variable region to transfer the antigen binding capacity of onevariable domain to another. Rather, it may only be necessary to transferthose residues that are necessary to maintain the activity of the targetbinding site. Given the explanations set forth in, e.g., U.S. Pat. Nos.5,585,089, 5,693,761, 5,693,762, and 6,180,370, it will be well withinthe competence of those skilled in the art, either by carrying outroutine experimentation or by trial and error testing to obtain afunctional engineered or humanized antibody.

As used herein the term “properly folded polypeptide” includespolypeptides (e.g., antibodies) in which all of the functional domainscomprising the polypeptide are distinctly active. As used herein, theterm “improperly folded polypeptide” includes polypeptides in which atleast one of the functional domains of the polypeptide is not active. Inone embodiment, a properly folded polypeptide comprises polypeptidechains linked by at least one disulfide bond and, conversely, animproperly folded polypeptide comprises polypeptide chains not linked byat least one disulfide bond.

As used herein the term “engineered” includes manipulation ofpolypeptide molecules by synthetic means (e.g. by recombinanttechniques, in vitro peptide synthesis, by enzymatic or chemicalcoupling of peptides or some combination of these techniques).

As used herein, the terms “linked,” “fused” or “fusion” are usedinterchangeably. These terms refer to the joining together of two moreelements or components, by whatever means including chemical conjugationor recombinant means. For example, a fusion protein can comprise a serumalbumin polypeptide, such as human serum albumin, and a secondpolypeptide. A fusion protein can also comprise an antibody Fc regionfused to a second polypeptide.

In the context of polypeptides, a “linear sequence” or a “sequence” isan order of amino acids in a polypeptide in an amino to carboxylterminal direction in which residues that neighbor each other in thesequence are contiguous in the primary structure of the polypeptide.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, an RNA or polypeptide. The processincludes any manifestation of the functional presence of the gene withinthe cell including, without limitation, gene knockdown as well as bothtransient expression and stable expression. It includes withoutlimitation transcription of the gene into messenger RNA (mRNA), transferRNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) orany other RNA product, and the translation of such mRNA intopolypeptide(s). If the final desired product is a biochemical,expression includes the creation of that biochemical and any precursors.Expression of a gene produces a “gene product.” As used herein, a geneproduct can be either a nucleic acid, e.g., a messenger RNA produced bytranscription of a gene, or a polypeptide which is translated from atranscript. Gene products described herein further include nucleic acidswith post transcriptional modifications, e.g., polyadenylation, orpolypeptides with post translational modifications, e.g., methylation,glycosylation, the addition of lipids, association with other proteinsubunits, proteolytic cleavage, and the like.

As used herein, an inhibitor of cysteine degradation is a molecule thatminimizes or prevents the breakdown of cysteine. Examples of inhibitorsof cysteine degradation include, but are not limited to, antioxidants,organic acids, organic aldehydes, and unsaturated lipids, specificallycompounds such as glutathione, pyruvate or pyruvate-containing molecules(e.g., methyl pyruvate or ethyl pyruvate), glyceraldehyde, glyoxylicacid, or citrate. In certain embodiments, antioxidants have one or morefree thiol groups. Exemplary antioxidants include, but are not limitedto glutathione, penicillamine, N-acetyl-cysteine, ascorbic acid, lipoicacid, carotenes, alpha-tocopherol, and ubiquinol. The most commonorganic acids are the carboxylic acids, although sulfonic acids can alsobe used. Examples of organic acids include, but are not limited to,lactic acid, formic acid, citric acid, oxalic acid, and uric acid.Examples of organic aldehydes include, but are not limited to methanal(formaldehyde), ethanal (acetaldehyde), propanal (propionaldehydc), andbutanal (butyraldehyde). Unsaturated lipids are lipids or fatty acidsthat contain one or more carbon-carbon double bonds in the lipid chain.Useful unsaturated lipids that can be used to inhibit cysteinedegradation include, but are not limited to, linoleic acid, linolenicacid, stearic acid, arachidonic acid, palmitic acid, docosahexaenoicacid, oleic acid, myristoleic acid, palmitoleic acid, elaidic acid,erucic acid, and vaccenic acid.

The concentration in which an inhibitor of cysteine degradation iscapable of reducing trisulfide formation can vary depending on theinhibitor chosen. Generally, the inhibitor will included in the culturemedium at a concentration of about_50 μM to about 500 mM. In certainembodiments the inhibitor is added at a concentration of about 100 μM toabout 100 mM or at a concentration of about 1 mM to 100 mM or at aconcentration of about 5 mM to about 50 mM or at a concentration ofabout 5 mM to about 30 mM or at a concentration of about 10 mM to about20 mM or at a concentration of about 15 mM to about 20 mM. In someembodiments, the ratio of the concentration of the inhibitor to theconcentration of cysteine present (e.g., in a cell culture medium) canbe from about 100:1 to about 1:10, about 20:1 to 1:10, or about 10:1 to1:10. In some embodiments, the ratio of the concentration of theinhibitor to the concentration of cysteine can be from about 5:1 toabout 1:10. In some embodiments, the ratio of the concentration of theinhibitor to the concentration of cysteine can be from about 5:1 toabout 1:5, 5:1 to 1:4, 5:1 to 1:3, or 5:1 to 1:2.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the development or spread ofcancer. Beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,domestic animals, farm animals, and zoo, sports, or pet animals such asdogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, andso on.

As used herein, phrases such as “a subject that would benefit fromadministration of a binding molecule” and “an animal in need oftreatment” includes subjects, such as mammalian subjects, that wouldbenefit from administration of a binding molecule used, e.g., fordetection of an antigen recognized by a binding molecule (e.g., for adiagnostic procedure) and/or from treatment, i.e., palliation orprevention of a disease such as cancer, with a binding molecule whichspecifically binds a given target protein. As described in more detailherein, the binding molecule can be used in unconjugated form or can beconjugated, e.g., to a drug, prodrug, or an isotope.

II. Trisulfide Bonds

“Trisulfide bonds” are generated by the insertion of an additionalsulfur atom into a disulfide bond, thereby resulting in the covalentbonding of three consecutive sulfur atoms. Trisulfide bonds and methodsof preventing and removing trisulfide bonds have been discussed inInternational Publication No. WO 2011/041721 and U.S. ProvisionalApplication Nos. 61/485,973 (filed May 13, 2011) and 61/617,529 (filedMar. 29, 2012), each of which is herein incorporated by reference in itsentirety. Trisulfide bonds can form between cysteine residues inproteins and can form intramolecularly (i.e., between two cysteines inthe same protein) or intermolecularly (i.e. between two cysteines inseparate proteins). In the case of antibodies, such as IgG1 antibodies,two intermolecular disulfide bonds link the heavy chains together and anintermolecular disulfide bonds also links each of the heavy and lightchains. Similarly, IgG2 molecules contain three intermolecular disulfidebonds that link the heavy chains, and IgG3 molecules contain 6-16intermolecular disulfide bonds that link the heavy chains. Trisulfidemodifications can occur at either of these disulfide linkages, but occurmore frequently at the heavy-light (HL) link than at the heavy-heavy(HH) link.

In some embodiments, trisulfide bonds decrease storage stability. Inother embodiments, trisulfide bonds increase protein aggregation. Instill other embodiments, trisulfide bonds increase protein oxidation,such as increasing methionine oxidation, potentially resulting indisassociation of antibody polypeptide chains (e.g. H-L and/or H-Hchains).

The presence of trisulfide bonds can be detected using any of a numberof methods, including methods described herein and methods known andavailable now or in the future to those of skill in the art. Forexample, trisulfide bonds can be detected using peptide mapping and canbe detected based on an increase in mass of the intact protein due to anextra sulfur atom (32 Da). Trisulfide bonds can be detected using massspectrum, or by high pressure liquid chromatography and massspectrometry (peptide mapping utilizing a LC-MS system). An informativeand quantitative analysis can be achieved through peptide mappingwherein select peptides derived from the intact molecule, includingthose containing sulfide bonds, are analyzed by LC-MS. In addition,trisulfide bonds can also be detected indirectly, e.g. by assessingmolecular folding or thermal stability.

As described in more detail herein, the presence of trisulfide bonds inantibodies can be detected or identified as a result of increasedsensitivity to heat treatment, for example as demonstrated by anincreased level of fragmentation following sample preparation fornon-reducing electrophoresis.

In some embodiments, the heat treatment can be at a temperature of atleast about 40° C., at least about 50° C., at least about 60° C., atleast about 70° C., at least about 80° C., at least about 90° C., atleast about 95° C., or at least about 100° C. In some embodiments, theheat treatment can be at a temperature of about 40° C., about 50° C.,about 60° C., about 70° C., about 80° C., about 90° C., or about 100° C.In some embodiments, the heat treatment can be at a temperature of lessthan about 120° C. In some embodiments, the heat treatment can be atemperature of about 60° C. to about 100° C.

According to the methods described herein, the heat treatment can beperformed in the presence of an alkylating agent, such as n-ethylmaleimide (NEM) or iodoacetamide. The concentration of the alkylatingagent such as NEM or iodoacetamide can be about 0.5 mM, about 1 mM,about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM,about 8 mM, about 9 mM, about 10 mM, or about 15 mM. The concentrationof the alkylating agent such as NEM or iodoacetamide can also be fromabout 0.5 mM to about 15 mM, from about 1.0 mM, to about 15 mM, fromabout 0.5 mM to about 10 mM, or from about 1.0 mM to about 10 mM.

According to the methods described herein, the heat treatment can lastfor a variable period of time. For example, the protein can be heatedfor about 30 seconds, about 1 minute, about 2 minutes, about 3 minutes,about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes,about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes,about 20 minutes, or about 30 minutes. In some embodiments, the proteinis heated from about 30 seconds to about 10 minutes.

In one particular embodiment described herein, the protein is heated forabout 5 minutes at a temperature of about 100° C. in the presence ofabout 5 mM NEM.

According to the methods described herein, the non-reducingelectrophoresis can be, for example, capillary electrophoresis,microfluidic chip electrophoresis (e.g. LC-90 assay), or polyacrylamidegel electrophoresis.

The quantity of trisulfide bonds can be reported in several differentways. For example, the “percentage of trisulfide bonds” present in asample could represent the number of trisulfide bonds among the totalnumber of all sulfide bonds present (i.e., disulfide and trisulfidebonds). However, unless otherwise stated, trisulfide bonds are reportedherein as the percentage of proteins in a sample that contain at leastone trisulfide bond. Thus, if only one protein in a sample of 100proteins contains a single trisulfide bond, as defined herein, 1% of theprotein in the sample contains trisulfide bonds. Similarly, if only twoproteins in a sample of 100 proteins each contain one or more trisulfidebonds, as defined herein, 2% of the proteins in the sample containtrisulfide bonds. Moreover, if only one protein in a sample of 100proteins contains 5 trisulfide bonds, as defined herein, 1% of theprotein in the sample contains trisulfide bonds (i.e., one trisulfidebond-containing protein in a sample of 100 proteins).

According to the methods described herein at least 1, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 15,at least 20, at least 30, at least 40, at least 50, at least 60, or atleast 70 percent of the proteins in a sample can contain trisulfidebonds. In another embodiment, from about 5 to about 50, from about 5 toabout 30, from about 5 to about 20, or from about 5 to about 15 percentof proteins in a sample can contain trisulfide bonds. In anotherembodiment, from about 10 to about 50, from about 10 to about 30, fromabout 10 to about 20, or from about 10 to about 15 percent of theproteins in a protein sample can contain trisulfide bonds.

In addition, it can be useful to determine the percentage of proteinscontaining trisulfide bonds in a sample after exposing the sample ofproteins to free cysteine to determine the efficacy of the conversion oftrisulfide bonds to disulfide bonds. Thus, for example, in someembodiments, after exposure to free cysteine, less than about 10, lessthan about 5, less than about 3, less than about 2, or less than about 1percent of proteins in the sample contain trisulfide bonds.

The number of trisulfide bonds in a sample can be decreased according tothe methods described herein. This decrease can be the result ofconversion of trisulfide bonds to disulfide bonds and/or in theelimination of both trisulfide and disulfide bonds. Thus, in someembodiments the percentage of proteins containing trisulfide bonds in asample is decreased by at least about 25%, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, or about 100%. In addition, insome embodiments of the methods described herein, the percentage ofproteins containing trisulfide bonds converted to disulfide bonds is atleast about 25%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, or about 100%.

In some embodiments, subsequent to exposing proteins to a solutioncomprising a reducing agent, less than about 10%, about 5%, or about 1%of the proteins contain trisulfide bonds.

In some embodiments, the methods herein provide compositions ofantibodies or fragments thereof wherein about Y % to about Z % of theantibodies or fragments thereof in the composition comprise one or moretrisulfide bonds, wherein Y % and Z % are any range of values selectedfrom the group of values represented in following table, as indicated by“Y/Z” wherein the lower “Y” value is selected from the corresponding rowand the upper “Z” value is selected from the corresponding column:

% 0.05 0.1 0.5 1 2 3 4 5 6 7 8 9 10 12 15 0.01 Y/Z Y/Z Y/Z Y/Z Y/Z Y/ZY/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z 0.05 Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/ZY/Z Y/Z Y/Z Y/Z Y/Z Y/Z 0.1 Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/ZY/Z Y/Z 0.5 Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z 1 Y/Z Y/ZY/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z 2 Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/ZY/Z Y/Z 3 Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z 4 Y/Z Y/Z Y/Z Y/Z Y/Z Y/ZY/Z Y/Z 5 Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z 6 Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z 7 Y/ZY/Z Y/Z Y/Z Y/Z 8 Y/Z Y/Z Y/Z Y/Z 9 Y/Z Y/Z Y/Z 10 Y/Z Y/Z 12 Y/Z

In some embodiments, the methods herein provide compositions ofantibodies or fragments thereof, wherein about Y % to about Z % of theantibodies or fragments thereof in said composition comprise onlydisulfide bonds, wherein Y % and Z % are any range of values selectedfrom group of values represented in the following table, as indicated by“Y/Z” wherein the lower “Y” value is selected from the corresponding rowand the upper “Z” value is selected from the corresponding column:

% 65 70 75 80 85 90 95 97 98 99 99.5 99.9 60 Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/ZY/Z Y/Z Y/Z Y/Z Y/Z 65 Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z 70Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z 75 Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/ZY/Z Y/Z 80 Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z 85 Y/Z Y/Z Y/Z Y/Z Y/Z Y/ZY/Z 90 Y/Z Y/Z Y/Z Y/Z Y/Z Y/Z 95 Y/Z Y/Z Y/Z Y/Z Y/Z 97 Y/Z Y/Z Y/Z Y/Z98 Y/Z Y/Z Y/Z 99 Y/Z Y/Z 99.5 Y/Z

III. Reducing Agent Treatment

According to the methods described herein, trisulfide bonds can bereduced by (a) applying a protein sample containing trisulfide bonds toa chromatographic medium; and (b) contacting a solution comprising areducing agent with the protein in association with the chromatographicmedium.

The protein sample can be any sample containing a protein withtrisulfide bonds that is amenable to application to a chromatographicmedium. For example, the protein sample can be a biological sample.Thus, the sample can be isolated from a patient, a subject, or a modelorganism. The sample can also be isolated from a cell culture and canbe, for example, a sample obtained from a cell lysate or a sampleobtained from cell supernatant. In addition, the sample can be acommercially available protein preparation (such as, for example,commercially available antibody preparations).

The protein in the sample can be a recombinant protein, a syntheticprotein, or a naturally occurring protein.

The chromatographic medium can be any chromatographic medium known inthe art now or in the future. The chromatographic medium can be one towhich the protein in the protein sample is bound, i.e. a chromatographicmedium that does not operate in a flow-through mode. Binding of theprotein can provide certain advantages, for example, by limiting motionof the protein and thereby preventing the formation of undesirabledisulfide bonds. For example, the chromatographic medium can be an ionex change chromatography medium, a mixed mode chromatography medium, anaffinity chromatography medium, a pseudo-affinity chromatography medium.The affinity chromatography medium can be, for example a lectinchromatography medium, a metal binding chromatography medium such as anickel chromatography medium, a GST chromatography medium, a Protein Gchromatography medium, a Protein A chromatography medium, or animmunoaffinity chromatography medium. The chromatographic medium can bein the form of a column, a chromatography resin, a similar bindingmatrix in another format such as a 96-well format. In addition, theprotein sample can be bound to a suitably modified membrane. In aparticular embodiment, the chromatography medium is a protein A affinitychromatography medium. In another particular embodiment, thechromatography medium is an antibody Fc region-binding chromatographymedium.

The solution comprising the reducing agent can be any solution that iscompatible with the chromatography medium and desired proteinfunction/biological activity (e.g., a solution that does not disrupt theintegrity of the column or irreversibly denature or inactivate thetarget protein). Similarly, the reducing agent can be any reducing agentthat is compatible with the chromatography medium and desired proteinfunction/biological activity. For example, the reducing agent can becysteine (including, for example, L-cysteine and N-acetyl cysteine),cysteine hydrochloride, cysteamine, sulfur dioxide, hydrogen sulfide,glutathione (GSH) (including, for example, L-glutathione (L-GSH),thioglycolic acid, bisulfite, ascorbic acid, sorbic acid, TCEP(tris(2-carboxyethyl)phosphine) and/or fumaric acid.

In some embodiments, the solution comprising the reducing agent furthercomprises additional components that decrease trisulfide bond levels. Insome embodiments, the solution comprising the reducing agent furthercomprises an alkali metal such as sodium, lithium, potassium, rubidium,or cesium. In some embodiments, the alkali metal is sodium phosphate.For example, the solution can be a phosphate buffered saline (PBS)solution. In some embodiments, the pH of the solution comprisingcysteine is a neutral pH (i.e., about 7). In some embodiments, the pH ofthe solution comprising cysteine is in a range from about 3 to about 9,from about 4 to about 8, from about 5 to about 7, from about 6 to about7, from about 5 to about 9, from about 5 to about 8, and from about 6 toabout 8.

According to the methods described herein, the reducing agent can befree cysteine. The concentration of free cysteine in the solution canbe, for example, about 0.1 mM or more and less than about 10 mM. In someembodiments, the concentration of cysteine can be, for example, fromabout 0.5 mM to about 9 mM, from about 0.5 mM to about 8 mM, from about0.5 mM to about 7 mM, from about 0.5 mM to about 6 mM, from about 0.5 mMto about 5 mM, from 0.5 mM to about 4 mM, from about 0.5 mM to about 1.0mM, from about 0.5 mM to about 2.0 mM, from about 0.5 mM to about 3 mM,from about 0.5 mM to about 4 mM, from about 0.5 mM to about 5 mM. Insome embodiments, the concentration of cysteine is from about 1.0 mM toabout 9 mM, from about 1.0 mM to about 8 mM, from about 1.0 mM to about7 mM, from about 1.0 mM to about 6 mM, from about 1.0 mM to about 5 mM,from about 1.0 mM to about 4 mM, or from about 1.0 mM to about 3 mM. Insome particular embodiments, the concentration is about 1.0 mM cysteineor about 3.0 mM cysteine. In some embodiments, the cysteine isL-cysteine. In some embodiments, the cysteine is N-acetyl cysteine.

According to the methods described herein, the reducing agent can beglutathione. The concentration of glutathione in the solution can be,for example, about 0.1 mM or more and less than about 10 mM. In someembodiments, the concentration of glutathione can be, for example, fromabout 0.5 mM to about 9 mM, from about 0.5 mM to about 8 mM, from about0.5 mM to about 7 mM, from about 0.5 mM to about 6 mM, from about 0.5 mMto about 5 mM, from 0.5 mM to about 4 mM, from about 0.5 mM to about 1.0mM, from about 0.5 mM to about 2.0 mM, from about 0.5 mM to about 3 mM,from about 0.5 mM to about 4 mM, from about 0.5 mM to about 5 mM. Insome embodiments, the concentration of glutathione is from about 1.0 mMto about 9 mM, from about 1.0 mM to about 8 mM, from about 1.0 mM toabout 7 mM, from about 1.0 mM to about 6 mM, from about 1.0 mM to about5 mM, from about 1.0 mM to about 4 mM, or from about 1.0 mM to about 3mM. In some particular embodiments, the concentration is about 1.0 mMglutathione. In some embodiments, the glutathione is L-glutathione.

The solution comprising the reducing agent can be applied in a singlestep or in multiple steps. The solution comprising the reducing agentcan be applied at a constant concentration or as a continuous orstep-wise gradient of increasing or decreasing concentrations. In someembodiments, the contact time of the reducing agent with the proteinsample can be controlled by selecting an appropriate column flow rate.For example, higher flow rates and shorter contact times can be usedwith higher concentrations of the reducing agent.

In some embodiments, the contact time is at least about 5 minutes, about10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about50 minutes, about 60 minutes, about 90 minutes, about 2 hours, about 3hours, about 4 hours, or about 5 hours. In some embodiments, the contacttime is less than about 24 hours, less than about 20 hours, less thanabout 12 hours, or less than about 6 hours. In some embodiments, thecontact time is about 1 hour.

In some embodiments, the linear flow velocity is at least about 10cm/hr, at least about 20 cm/hr, at least about 30 cm/hr, at least about40 cm/hr, at least about 50 cm/hr, at least about 60 cm/hr, at leastabout 70 cm/hr, at least about 80 cm/hr, at least about 90 cm/hr, atleast about 100 cm/hr, at least about 150 cm/hr, at least about 200cm/hr, at least about 250 cm/hr, at least about 300 cm/hr, at leastabout 350 cm/hr, at least about 400 cm/hr, at least about 450 cm/hr, atleast about 500 cm/hr, at least about 550 cm/hr. In some embodiments,the linear flow velocity is less than about 1000 cm/hr or less thanabout 750 cm/hr. In some embodiments, the linear flow velocity is avelocity that is compatible with large scale column purification ofantibodies. In some embodiments, the linear flow velocity is about 100cm/hr.

The application of a solution comprising the reducing agent to theprotein sample in association with a chromatographic medium can bepreceded by, combined with, or followed by additional wash steps. Forexample, a PBS wash can be applied before the solution comprising thereducing agent is applied to the chromatographic medium. The wash can beused, for example, to clear impurities. A wash with a high salt buffersolution can also be used to promote the clearance of impurities, forexample, after the solution comprising the reducing agent is applied tothe chromatographic medium. A high salt wash can also be combined withthe reducing agent wash, thereby reducing the total number of washesrequired. Additionally and/or alternatively, a reducing-agent-free washcan be applied after the reducing agent wash to remove the reducingagent from the protein while the protein is still on the column.Additionally and/or alternatively, a low salt concentration wash can beapplied to promote efficient elution during a subsequent elution step.

After the application of a solution comprising a reducing agent to thechromatographic medium, and optionally additional wash steps, theprotein can be eluted from the chromatographic medium. Elution can beperformed using any technique known in the art, for example, when usinga Protein A chromatographic medium elution can be performed by applyinga low pH solution (e.g., a pH of about 3.5). The pH of the eluted samplecan then be adjusted to a more neutral pH (e.g., a pH of about 5 to 9,pH of about 6 to 8, or pH of about 7) using a basic solution.

IV. Treatment with Inhibitor of Cysteine Degradation

According to the methods described herein, trisulfide bonds can bereduced using inhibitors of cysteine degradation. For example, culturingcells expressing proteins in the presence of an inhibitor of cysteinedegradation can reduce trisulfide bonds.

In some embodiments, the solution comprising the inhibitor of cysteinedegradation further is a cell-culture medium. In some embodiments, thecell-culture medium is a common cell-culture medium, for example,Iscove's Modified Dulbecco's Medium (IMDM) or Dulbecco's Modified EagleMedium (DMEM). In some embodiments, the cell-culture medium furthercomprises glucose. In some embodiments, the cell-culture mediumcomprises cysteine. In some embodiments, a medium can contain glucoseand cysteine. In some embodiments, a medium can contain a solution ofamino acids and cysteine.

In some embodiments, the ratio of the concentration of the inhibitor tothe concentration of cysteine present in a cell culture medium can be,for example, from about 100:1 to about 1:10, about 20:1 to 1:10, orabout 10:1 to 1:10. In some embodiments, the ratio of the concentrationof the inhibitor to the concentration of cysteine can be from about 5:1to about 1:10. In some embodiments, the ratio is about 1:1. In someembodiments, the ratio of the concentration of the inhibitor to theconcentration of cysteine can be from about 5:1 to about 1:5, 5:1 to1:4, 5:1 to 1:3, or 5:1 to 1:2.

According to the methods described herein, the inhibitor of cysteinedegradation can be pyruvate or pyruvate-containing molecules. Theconcentration of pyruvate or pyruvate-containing molecules can be, forexample, about 50 μM to about 500 mM. In some embodiments, theconcentration of pyruvate or pyruvate-containing molecules can be, forexample, from about 1 mM to about 250 mM, from about 1 mM to about 100mM, from about 1 mM to about 50 mM, from about 1 mM to about 30 mM, fromabout 1 mM to about 25 mM, or from about 1 mM to about 20 mM. In someembodiments, the concentration of pyruvate or pyruvate-containingmolecules can be, for example, from about 5 mM to about 250 mM, fromabout 5 mM to about 100 mM, from about 5 mM to about 50 mM, from about 5mM to about 30 mM, from about 5 mM to about 25 mM, or from about 5 mM toabout 20 mM. In some embodiments, the concentration of pyruvate orpyruvate-containing molecules can be, for example, from about 10 mM toabout 250 mM, from about 10 mM to about 100 mM, from about 10 mM toabout 50 mM, from about 10 mM to about 30 mM, from about 10 mM to about25 mM, or from about 10 mM to about 20 mM. In some embodiments, theconcentration of pyruvate or pyruvate-containing molecules can be, forexample, from about 15 mM to about 250 mM, from about 15 mM to about 100mM, from about 15 mM to about 50 mM, from about 15 mM to about 30 mM,from about 15 mM to about 25 mM, or from about 15 mM to about 20 mM. Insome embodiments, the pyruvate-containing molecule can be, for example,methyl pyruvatc or ethyl pyruvate. In some embodiments, the inhibitor ofcysteine degradation is pyruvate.

According to the methods described herein, the inhibitor of cysteinedegradation can be glyceraldehyde. The concentration of glyceraldehydecan be, for example, about 50 μM to about 500 mM. In some embodiments,the concentration of glyceraldehyde can be, for example, from about 1 mMto about 250 mM, from about 1 mM to about 100 mM, from about 1 mM toabout 50 mM, from about 1 mM to about 30 mM, from about 1 mM to about 25mM, or from about 1 mM to about 20 mM. In some embodiments, theconcentration of glyceraldehyde can be, for example, from about 5 mM toabout 250 mM, from about 5 mM to about 100 mM, from about 5 mM to about50 mM, from about 5 mM to about 30 mM, from about 5 mM to about 25 mM,or from about 5 mM to about 20 mM. In some embodiments, theconcentration of glyceraldehyde can be, for example, from about 10 mM toabout 250 mM, from about 10 mM to about 100 mM, from about 10 mM toabout 50 mM, from about 10 mM to about 30 mM, from about 10 mM to about25 mM, or from about 10 mM to about 20 mM. In some embodiments, theconcentration of glyceraldehyde can be, for example, from about 15 mM toabout 250 mM, from about 15 mM to about 100 mM, from about 15 mM toabout 50 mM, from about 15 mM to about 30 mM, from about 15 mM to about25 mM, or from about 15 mM to about 20 mM.

According to the methods described herein, the inhibitor of cysteinedegradation can be glyoxylic acid. The concentration of glyoxylic acidcan be, for example, about 50 μM to about 500 mM. In some embodiments,the concentration of glyoxylic acid can be, for example, from about 1 mMto about 250 mM, from about 1 mM to about 100 mM, from about 1 mM toabout 50 mM, from about 1 mM to about 30 mM, from about 1 mM to about 25mM, or from about 1 mM to about 20 mM. In some embodiments, theconcentration of glyoxylic acid can be, for example, from about 5 mM toabout 250 mM, from about 5 mM to about 100 mM, from about 5 mM to about50 mM, from about 5 mM to about 30 mM, from about 5 mM to about 25 mM,or from about 5 mM to about 20 mM. In some embodiments, theconcentration of glyoxylic acid can be, for example, from about 10 mM toabout 250 mM, from about 10 mM to about 100 mM, from about 10 mM toabout 50 mM, from about 10 mM to about 30 mM, from about 10 mM to about25 mM, or from about 10 mM to about 20 mM. In some embodiments, theconcentration of glyoxylic acid can be, for example, from about 15 mM toabout 250 mM, from about 15 mM to about 100 mM, from about 15 mM toabout 50 mM, from about 15 mM to about 30 mM, from about 15 mM to about25 mM, or from about 15 mM to about 20 mM.

In some embodiments, protein trisulfide bond formation is prevented,inhibited or curtailed via monitoring and/or manipulating theconcentration of cysteine and/or cystine present in a cell culture mediaat or below a threshold level. For example, in order to decrease ormaintain low cysteine and/or cystine levels, methionine can replacecysteine and/or cystine in a nutrient feed. In some embodiments, thetotal amount of feed can be lowered in order to reduce cysteine and/orcystine levels. The threshold level can be, for example, about 150 μM,about 125 μM, about 100 μM, about 90 μM, about 80 μM, about 70 μM, about60 μM, about 50 μM, about 40 μM, about 30 μM, or about 20 μM. Thethreshold level can be, for example, about 10 mM, about 9 mM, about 8mM, about 7 mM, about 6 mM, about 5 mM, about 4 mM, about 3 mM, about 2mM, about 1 mM, about 0.9 mM, about 0.8 mM, about 0.7 mM, about 0.6 mM,about 0.5 mM, about 0.4 mM, about 0.3 mM, about 0.2 mM, or about 0.1 mM.

In some embodiments, protein trisulfide bond formation is prevented,inhibited or curtailed via monitoring and/or manipulating theconcentration of hydrogen sulfide (H₂S) at or below a threshold level.The threshold level can be, for example, about 10 parts per million(ppm), about 5 ppm, about 4 ppm, about 3 ppm, about 2 ppm, about 1 ppm,about 0.9 ppm, about 0.8 ppm, about 0.7 ppm, about 0.6 ppm, about 0.5ppm, about 0.4 ppm, about 0.3 ppm, about 0.2 ppm, or about 0.1 ppm. Insome embodiments, the threshold levels is about 500 parts per billion(ppb), about 250 ppb, about 200 ppb about 100 ppb, or about 50 ppb. Insome embodiments, the use of an inhibitor of cysteine degradationmaintains the H₂S concentration at a level of below about 10 ppm, about5 ppm, about 4 ppm, about 3 ppm, about 2 ppm, about 1 ppm, about 0.9ppm, about 0.8 ppm, about 0.7 ppm, about 0.6 ppm, about 0.5 ppm, about0.4 ppm, about 0.3 ppm, about 0.2 ppm, or about 0.1 ppm. In someembodiments, the use of an inhibitor of cysteine degradation maintainsthe H₂S concentration at a level of below about 500 ppb, about 250 ppb,about 200 ppb about 100 ppb, or about 50 ppb

In some embodiments, addition of inhibitor of cysteine degradationreduces release of hydrogen sulfide by at least 55%, 60%, 65%, 70%, 75%,or 80% compared to absence of inhibitor.

V. Cell Culture

According to the methods described herein, the trisulfidebond-containing protein can be a protein that is produced in a cellculture. The protein produced by the cell culture can be a recombinantprotein or a protein naturally expressed by the cells grown in theculture. The protein can be an intracellular protein, a transmembraneprotein, or a secreted protein.

In some particular embodiments, the protein is an antibody. For example,the antibody can be an antibody that binds to LINGO-1. The antibody canalso be an antibody that binds to human LINGO-1. In some embodiments theanti-LINGO-1 antibody is selected from the group consisting of Li13,Li33, Li62, Li81, and Li113 and chimeric or humanized versions thereof.In some embodiments, the anti-LINGO-1 antibody is a variant of Li13,Li33, Li62, Li81, or Li113. For example, in the some embodiments, theanti-LINGO-1 antibody is Li81 M96L or Li81 M96F, each of which containan amino acid substitution in methionine 96 of the light chain of theLi81 antibody. “Mab1” and “mAb-A” as referred to in the examples beloware the anti-LINGO-1 antibody Li81.

Li13 and Li33 are monoclonal antibody Fab fragments that were identifiedand isolated from phage display libraries as described in Hoet et al.,Nat. Biotech. 23:344-348 (2005); Rauchenberger, et al., J. Biol. Chem.278:194-205 (2003); and Knappik, et al., J. Mol. Biol. 296:57-86 (2000),all of which are incorporated herein by reference in their entireties.In these experiments, the MorphoSys Fab-phage display library HuCAL®GOLD, which comprises humanized synthetic antibody variable regions wasscreened against recombinant human soluble LINGO-1-Fc protein bystandard ELISA AND IHC screening methods. Fab-phages that specificallybound to LINGO-1 were purified and characterized. Li13 and Li33 aredescribed in more detail in International Published Application WO2007/008547, which is incorporated herein by reference in its entirety.

Antibody Li81 is derived from Li13 and Li33. It includes the Li13 lightchain and an affinity matured heavy chain. Li81 is described in moredetail in International Published Application WO 2008/086006, which isincorporated herein by reference in its entirety. Li81 M96L and Li81M96F are described in WO 2010/005570, which is incorporated herein byreference in its entirety.

Li62 is derived from Li33. It includes the Li33 heavy chain and a lightchain that was identified in a library screen. Li113 was identified byisolated targeted phage display. It includes the Li62 light chain aswell as the Li62 VH CDR1 and CDR2 sequences, but has alterations in theVH CDR3 sequence. Li62 and Li113 are described in InternationalApplication No. PCT/US2009/003999 (published as WO 2010/005570), whichis incorporated herein by reference in its entirety.

The VH sequences of Li13, Li33, Li62, Li81, and Li113 are shown in Table1 below.

TABLE 1 LINGO-1 Antibody VH Sequences Anti- VH VH body VH SEQUENCE CDR 1VH CDR2 CDR3 Li13 EVQLLESGGGLVQPGGSLRLSCAASGF HYEMY RIVSSGG EGDNDTFSHYEMYWVRQAPGKGLEWVSRIVS (SEQ ID FTKYAD AFDISGGFTKYADSVKGRFTISRDNSKNTLY NO: 2) SVKG (SEQ IDLQMNSLRAEDTAVYYCATEGDNDAFD (SEQ ID NO: 4) IWGQGTTVTVSS (SEQ ID NO: 1)NO: 3) Li33 EVQLLESGGGLVQPGGSLRLSCAASGF IYPMF WIGPSG EGHNDTFSIYPMFWVRQAPGKGLEWVSWIGPS (SEQ ID GITKYA WYFDLGGITKYADSVKGRFTISRDNSKNTLYL NO: 5) DSVKG (SEQ IDQMNSLRAEDTATYYCAREGHNDWYF (SEQ ID NO: 7) DLWGRGTLVTVSS (SEQ ID NO: 97)NO: 6) Li62 EVQLLESGGGLVQPGGSLRLSCAASGF IYPMF WIGPSG EGHNDTFSIYPMFWVRQAPGKGLEWVSWIGPS (SEQ ID GITKYA WYFDLGGITKYADSVKGRFTISRDNSKNTLYL NO: 9) DSVKG (SEQ IDQMNSLRAEDTATYYCAREGHNDWYF (SEQ ID NO: 11) DLWGRGTLVTVSS (SEQ ID NO: 8)NO: 10) Li81 EVQLLESGGGLVQPGGSLRLSCAASGF AYEMK VIGPSGG EGDNDTFSAYEMKWVRQAPGKGLEWVSVIGP (SEQ ID FTFYADS AFDISGGFTFYADSVKGRFTISRDNSKNTLY NO: 13) VKG (SEQ IDLQMNSLRAEDTAVYYCATEGDNDAFD (SEQ ID NO: 15) IWGQGTTVTVSS (SEQ ID NO: 12)NO: 14) Li113 EVQLLESGGGLVQPGGSLRLSCAASGF IYPMF WIGPSG EGTYDTFSIYPMFWVRQAPGKGLEWVSWIGPS (SEQ ID GITKYA WYLDLGGITKYADSVKGRFTISRDNSKNTLYL NO: 17) DSVKG (SEQ IDQMNSLRAEDTATYYCAREGTYDWYL (SEQ ID NO: 19) DLWGRGTLVTVSS (SEQ ID NO: 16)NO: 18)

The VL sequences of Li13, Li33, Li62, Li81, Li113, Li81 M96L, and Li81M96F are shown in the Table 2 below.

TABLE 2 LINGO-1 Antibody VL Sequences Anti- VL VL VL body VL SEQUENCECDR1 CDR2 CDR3 Li13 DIQMTQSPATLSLSPGERATLSCRAS RASQS DASNR QQRSNQSVSSYLAWYQQKPGQAPRLLIYDA VSSYL AT (SEQ WPMYTSNRATGIPARFSGSGSGTDFTLTISSLE A ID (SEQ ID PEDFAVYYCQQRSNWPMYTFGQGT(SEQ ID NO: 22) NO: 23) KLEIK (SEQ ID NO: 20) NO: 21) Li33DIQMTQSPGTLSLSPGERATLSCRAS RASQS DASNR QQYDK QSVSSYLAWYQQKPGQAPRLLIYDAVSSYL AT WPLT SNRATGIPARFSGSGSGTEFTLTISSLQ A (SEQ (SEQ ID (SEQ IDSEDFAVYYCQQYDKWPLTFGGGTK ID NO: 26) NO: 27) VEIK (SEQ ID NO: 24) NO: 25)Li62 DIQMTQSPSFLSASVGDSVAITCRAS RASQD DASNL QQYDTQDISRYLAWYQQRPGKAPKLLIYDA ISRYL QT (SEQ LHPS SNLQTGVPSRFSGSGSGTDFTFTITSLA (SEQ ID (SEQ ID QPEDFGTYYCQQYDTLHPSFGPGTTV ID NO: 30) NO: 31)DIK (SEQ ID NO: 28) NO: 29) Li81 DIQMTQSPATLSLSPGERATLSCRAS RASQS DASNRQQRSN QSVSSYLAWYQQKPGQAPRLLIYDA VSSYL AT (SEQ WPMYTSNRATGIPARFSGSGSGTDFTLTISSLE A (SEQ ID NO: (SEQ IDPEDFAVYYCQQRSNWPMYTFGQGT ID 34) NO: 35) KLEIK (SEQ ID NO: 32) NO: 33)Li113 DIQMTQSPSFLSASVGDSVAITCRAS RASQD DASNL QQYDTQDISRYLAWYQQRPGKAPKLLIYDA ISRYL QT (SEQ LHPS SNLQTGVPSRFSGSGSGTDFTFTITSLA (SEQ ID (SEQ ID QPEDFGTYYCQQYDTLHPSFGPGTTV ID NO: 38) NO: 39)DIK (SEQ ID NO: 36) NO: 37) Li81 DIQMTQSPATLSLSPGERATLSCRAS RASQS DASNRQQRSN M96L QSVSSYLAWYQQKPGQAPRLLIYDA VSSYL AT (SEQ WPLYTSNRATGIPARFSGSGSGTDFTLTISSLE A (SEQ ID NO: (SEQ IDPEDFAVYYCQQRSNWPLYTFGQGTK ID 100) NO: 94) LEIK (SEQ ID NO: 93) NO: 98)Li81 DIQMTQSPATLSLSPGERATLSCRAS RASQS DASNR QQRSN M96FQSVSSYLAWYQQKPGQAPRLLIYDA VSSYL AT (SEQ WPFYTSNRATGIPARFSGSGSGTDFTLTISSLE A (SEQ ID NO: (SEQ IDPEDFAVYYCQQRSNWPFYTFGQGTK ID 101) NO: 96) LEIK (SEQ ID NO: 95) NO: 99)

In another example, the antibody can be an antibody that binds to Fn14.Fn14, a TWEAK receptor, is a growth factor-regulated immediate-earlyresponse gene that decreases cellular adhesion to the extracellularmatrix and reduces serum-stimulated growth and migration. Meighan-Manthaet al., J. Biol. Chem. 274:33166-33176 (1999). In some embodiments, theanti-Fn14 antibody is selected from the group consisting of P4A8, P3G5,and P2D3 and chimeric or humanized versions thereof. Anti-Fn14antibodies P4A8, P3G5, and P2D3 were raised in Fn14-deficient mice byadministration of CHO cells expressing human surface Fn14 and boostedwith Fn14-myc-His protein. These antibodies are described in more detailin International Application Number PCT/US2009/043382, filed on May 8,2009, which is incorporated herein by reference in its entirety.

The VH sequences of P4A8, P3G5, and P2D3 antibodies are shown in Table 3below.

TABLE 3 Fn14 Antibody VH Sequences Anti- VH VH VH body VH SEQUENCE CDR1CDR2 CDR3 P4A8 QVQLQQSGPEVVRPGVSVKISCKGS DYGMH VISTY AYYGGYTFTDYGMHWVKQSHAKSLEWI (SEQ ID NGYTN NLYYA GVISTYNGYTNYNQKFKGKATMTVNO: 41) YNQKF MDY DKSSSTAYMELARLTSEDSAIYYCA KG (SEQ IDRAYYGNLYYAMDYWGQGTSVTVS (SEQ ID NO: 43) S (SEQ ID NO: 40) NO: 42) P3G5QVQLQQSGPEVVRPGVSVKISCKGS DYGIH VISTY AYYG GYTFTDYGIHWVKQSHAKSLEWIG(SEQ ID NGYTN NLYYA VISTYNGYTNYNQKFKGKATMTVD NO: 45) YNQKF MDYKSSSTAYMELARLTSEDSAIYYCAR KG (SEQ ID AYYGNLYYAMDYWGQGTSVTVSS (SEQ IDNO: 47) (SEQ ID NO: 44) NO: 46) P2D3 QVSLKESGPGILQPSQTLSLTCSFSG TSGMGHIYWD RGPDY FSLSTSGMGVSWIRQPSGKGLEWLA VS (SEQ DDKRY YGYYPHIYWDDDKRYNPSLKSRLTISKDTS ID NPSLK MDY RNQVFLKITSVDTADTATYYCARRG NO: 49)S (SEQ (SEQ ID PDYYGYYPMDYWGQGTSVTVSS ID NO: 51) (SEQ ID NO: 48) NO: 50)huP4A8 QVQLVQSGAEVKKPGASVKVSCKG GYTFTD VISTY AYYG versionSGYTFTDYGMHWVRQAPGQGLEW YGMH NGYTN NLYYA 1 MGVISTYNGYTNYNQKFKGRVTMT(SEQ ID YNQKF MDY VDKSTSTAYMELRSLRSDDTAVYY NO: 53) KG (SEQ IDCARAYYGNLYYAMDYWGQGTLVT (SEQ ID NO: 55) VSS (SEQ ID NO: 52) NO: 54)huP4A8 QVQLVQSGAEVKKPGASVKVSCKG GYTFTD VISTY AYYG versionSGYTFTDYGMHWVRQAPGQGLEWI YGMH NGYTN NLYYA 2 GVISTYNGYTNYNQKFKGRATMTV(SEQ ID YNQKF MDY DKSTSTAYMELRSLRSDDTAVYYC NO: 57) KG (SEQ IDARAYYGNLYYAMDYWGQGTLVTV (SEQ ID NO: 59) SS (SEQ ID NO: 56) NO: 58)

The VL sequences of P4A8, P3G5, and P2D3 antibodies are shown in Table 4below.

TABLE 4 Fn14 Antibody VL Sequences Anti- VL VL VL body VL SEQUENCE CDR1CDR2 CDR3 P4A8 DIVLTQSPASLAVSLGQRATISCRAS RASKSV ASNLE QHSRELKSVSTSSYSYMHWYQQKPGQPPKL STSSYS S (SEQ PFT LIKYASNLESGVPARFSGSGSGTDFIYMH ID (SEQ ID LNIHPVEEEDAATYYCQHSRELPFT (SEQ ID NO: 62) NO: 63)FGSGTKLEIK (SEQ ID NO: 60) NO: 61) P3G5 DIVLTQSPASLAVSLGQRATISCRAN RANKSASNLE QHSREL KSVSTSSYSYMHWYQQKPGQPPKL VSTSSY S (SEQ PFTLIKYASNLESGVPARFSGSGSGTDFI SYMH ID (SEQ ID LNIHPVEEEDAATYYCQHSRELPFT(SEQ ID NO: 66) NO: 67) FGSGTKLEIK (SEQ ID NO: 64) NO: 65) P2D3DIVLTQSPASLAVSLGQRATISCRAS RASKSV TSNLE QHSREL KSVSTSSYSYMHWYQQKPGQPPKLSTSSYS S (SEQ PWT LIKYTSNLESGVPARFSGSGSGTDFI YMH ID (SEQ IDLNIHPVEEEDAATYYCQHSRELPWT (SEQ ID NO: 70) NO: 71)FGGGTKLEIK (SEQ ID NO: 68) NO: 69) Humanized DIVLTQSPASLAVSLGQRATISCRASRASKSV YASNL QHSREL P4A8 KSVSTSSYSYMHWYQQKPGQPPKL STSSYS ES PFTversion 1 LIKYASNLESGVPARFSGSGSGTDFS YMH (SEQ ID (SEQ IDLNIHPMEEDDTAMYFCQHSRELPFT (SEQ ID NO: 74) NO: 75)FGGGTKLEIK (SEQ ID NO: 72) NO: 73) Humanized DIVLTQSPASLAVSLGQRATISCRASRATISC YASNL QHSREL P4A8 KSVSTSSYSYMHWYQQKPGQPPKL RASKSV ES PFT versionLIKYASNLESGVPARFSGSGSGTDFI STSSYS (SEQ ID (SEQ ID 2LNIHPMEEDDTAMYFCQHSRELPFT YMH NO: 78) NO: 79) FGGGTKLEIK (SEQ ID NO: 76)(SEQ ID NO: 77) Humanized DIVLTQSPASLAVSLGQRATISCRAS RASKSV YASNL QHSRELP4A8 KSVSTSSYSYMHWYQQKPGQPPKL STSSYS ES PFT versionLIKYASNLESGVPARFSGSGSGTDFI YMH (SEQ ID (SEQ ID 3LNIHPMEEDDTATYYCQHSRELPFT (SEQ ID NO: 82) NO: 83)FGGGTKLEIK (SEQ ID NO: 80) NO: 81)

Heavy and light chain sequences of humanized P4A8 antibodies are shownin Table 5 below.

TABLE 5 Chain Sequences of Chimeric and Humanized P4A8 Antibodies ChainDescription Sequence Chimeric QVQLQQSGPEVVRPGVSVKISCKGSGYTFTDYGMHWVKQSHAP4A8 heavy KSLEWIGVISTYNGYTNYNQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCARAYYGNLYYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 84) ChimericDIVLTQSPASLAVSLGQRATISCRASKSVSTSSYSYMHWYQQKP P4A8 lightGQPPKLLIKYASNLESGVPARFSGSGSGTDFILNIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 85) HumanizedQVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYGMHWVRQAP P4A8-IgG1GQGLEWMGVISTYNGYTNYNQKFKGRVTMTVDKSTSTAYME H1 heavyLRSLRSDDTAVYYCARAYYGNLYYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 86) HumanizedQVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYGMHWVRQAP P4A8-IgG1GQGLEWIGVISTYNGYTNYNQKFKGRATMTVDKSTSTAYMEL H2 heavyRSLRSDDTAVYYCARAYYGNLYYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 87) HumanizedDIVLTQSPASLAVSLGQRATISCRASKSVSTSSYSYMHWYQQKP P4A8 L1GQPPKLLIKYASNLESGVPARFSGSGSGTDFSLNIHPMEEDDTA kappa lightMYFCQHSRELPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 88) HumanizedDIVLTQSPASLAVSLGQRATISCRASKSVSTSSYSYMHWYQQKP P4A8 L2GQPPKLLIKYASNLESGVPARFSGSGSGTDFILNIHPMEEDDTA kappa lightMYFCQHSRELPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 89) HumanizedDIVLTQSPASLAVSLGQRATISCRASKSVSTSSYSYMHWYQQKP P4A8 L3GQPPKLLIKYASNLESGVPARFSGSGSGTDFILNIHPMEEDDTAT kappa lightYYCQHSRELPFTEGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 90) HumanizedQVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYGMHWVRQAP P4A8-IgG4GQGLEWMGVISTYNGYTNYNQKFKGRVTMTVDKSTSTAYME H1 heavyLRSLRSDDTAVYYCARAYYGNLYYAMDYWGQGTLVTVSSAS aglyTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL 5228PTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS T299ANTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSAYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 91) HumanizedQVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYGMHWVRQAP P4A8-IgG1GQGLEWMGVISTYNGYTNYNQKFKGRVTMTVDKSTSTAYME H1 heavyLRSLRSDDTAVYYCARAYYGNLYYAMDYWGQGTLVTVSSAS aglyTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL T299ATSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSAYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:92)

The cell culture can comprise, for example, bacterial cells, yeast cellsor mammalian cells. Cell types that can be used according to the presentmethods include any mammalian cells that are capable of growing inculture, for example, CHO (Chinese Hamster Ovary) (including CHO-K1, CHODG44, and CHO DUXB11), VERO, HeLa, (human cervical carcinoma), CVI(monkey kidney line), (including COS and COS-7), BHK (baby hamsterkidney), MDCK, C127, PC12, HEK-293 (including HEK-293T and HEK-293E),PER C6, NSO, WI38, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mousefibroblast), HAK (hamster kidney line), SP2/O (mouse myeloma),P3x63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells),RAJI (human lymphocyte) and 293 (human kidney) cells. In some particularembodiments, the cells are CHO cells or a derivative thereof.

In some embodiments, protein trisulfide bond formation is prevented,inhibited or curtailed via monitoring and manipulating the quantityand/or rate of nutrient feed, amino acid, or other nutrient supplementsprovided to a cell culture during a bioreactor culture run.

Optimal cell culture media compositions vary according to the type ofcell culture being propagated. In some embodiments, the nutrient mediais a commercially available media. In some embodiments, the nutrientmedia contains e.g., inorganic salts, carbohydrates (e.g., sugars suchas glucose, galactose, maltose or fructose), amino acids, vitamins(e.g., B group vitamins (e.g., B12), vitamin A vitamin E, riboflavin,thiamine and biotin), fatty acids and lipids (e.g., cholesterol andsteroids), proteins and peptides (e.g., albumin, transferrin,fibronectin and fetuin), serum (e.g., compositions comprising albumins,growth factors and growth inhibitors, such as, fetal bovine serum,newborn calf serum and horse serum), trace elements (e.g., zinc, copper,selenium and tricarboxylic acid intermediates), hydrolysates (hydrolyzedproteins derived from plant or animal sources), and combinationsthereof. Examples of nutrient medias include, but are not limited to,basal media (e.g., MEM, DMEM, GMEM), complex media (RPMI 1640, IscovesDMEM, Leibovitz L-15, Leibovitz L-15, TC 100), serum free media (e.g.,CHO, Ham F10 and derivatives, Ham F12, DMEM/F12). Common buffers foundin nutrient media include PBS, Hanks BSS, Earles salts, DPBS, HBSS, andEBSS. Media for culturing mammalian cells are well known in the art andare available from, e.g., Sigma-Aldrich Corporation (St. Louis, Mo.),HyClone (Logan, Utah), Invitrogen Corporation (Carlsbad, Calif.),Cambrex Corporation (E. Rutherford, N.J.), JRH Biosciences (Lenexa,Kans.), Irvine Scientific (Santa Ana, Calif.), and others. Othercomponents found in nutrient media can include ascorbate, citrate,cysteine/cystine, glutamine, folic acid, glutathione, linoleic acid,linolenic acid, lipoic acid, oleic acid, palmitic acid,pyridoxal/pyridoxine, riboflavin, selenium, thiamine, and transferrin.In some embodiments the nutrient media is serum-free media, aprotein-free media, or a chemically defined media. One of skill in theart will recognize that there are modifications to nutrient media whichwould fall within the scope of this invention.

In some embodiments, protein trisulfide bond formation is prevented,inhibited or curtailed via monitoring and/or manipulating theconcentration of cysteine and/or cystine present in a cell culture mediaat or below a threshold level. For example, in order to decrease ormaintain low cysteine and/or cystine levels, methionine can replacecysteine and/or cystine in a nutrient feed. In some embodiments, thetotal amount of feed can be lowered in order to reduce cysteine and/orcystine levels. The threshold level can be, for example about 150 μM,about 125 μM, about 100 μM, about 90 μM, about 80 μM, about 70 μM, about60 μM, about 50 μM, about 40 μM, about 30 μM, or about 20 μM.

In other embodiments, protein trisulfide bond formation is prevented,inhibited or curtailed via monitoring and/or manipulating theconcentration of hydrogen sulfide (H₂S), sodium sulfide, and/or sodiumhydrogen sulfide at or below a threshold level. In some embodiments areactant, scavenging molecule, or “sponge” can be introduced into thecell culture medium. A “sponge” as used herein, is a molecule, compound,or material that can reduce, eliminate, or inhibit the ability of sulfuror sulfur-containing compounds to form trisulfide bonds. For example,sodium sulfite can be introduced to react with and eliminate H₂S. Thethreshold level can be, for example, about 10 mM, about 9 mM, about 8mM, about 7 mM, about 6 mM, about 5 mM, about 4 mM, about 3 mM, about 2mM, about 1 mM, about 0.9 mM, about 0.8 mM, about 0.7 mM, about 0.6 mM,about 0.5 mM, about 0.4 mM, about 0.3 mM, about 0.2 mM, or about 0.1 mM.

In some embodiments, protein trisulfide bond formation is prevented,inhibited, or curtailed by harvesting the conditioned cell culture mediafrom a cell culture during a particular phase of cell growth. Typicalcell culture growth curves include inoculation of nutrient media withthe starter cells followed by lag phase growth. The lag phase isfollowed by log phase growth of the culture, ultimately resulting in aplateau phase. As used herein, the term “bioreactor run” can include oneor more of the lag phase, log phase, or plateau phase growth periodsduring a cell culture cycle. Thus, in some embodiments, the proteinsamples are obtained from cell culture during maximum growth phase inorder to minimize formation or accumulation trisulfide bonds. In someembodiments, the cell culture is harvested when the cell culture is atapproximately maximum cell density. In another embodiment, proteinsamples are obtained from cell culture at a time when proteinproduction, e.g. recombinant protein production, is maximal. Forexample, in some embodiments, harvest occurs on day 13 of a bioreactorrun. In some embodiments, the cell culture is harvested prior to thepeak induction of trisulfide bond formation.

In some embodiments, protein trisulfide bond formation is prevented,inhibited or curtailed via temporarily or constantly growing the cellculture at a preferred temperature. In some embodiments, the temperatureis maintained at a temperature of above 35° C. In other words, thetemperature can be maintained at a steady temperature throughout theculture, or the temperature can be shifted. For example, the cellculture can be maintained at about 35° C. throughout the culturingprocess. In addition, the cell culture can be grown at 35° C. butshifted to a lower temperature, e.g., a temperature of about 32° C. Thetemperature shift can occur, for example, 1, 2, 3, 4, or 5 days beforeharvesting the culture. For example, if harvest occurs on day 13 of abioreactor process, the temperature shift can occur, for example, at day9 of the bioreactor process. In order to compensate for slower growth,and associated excess feed at lower temperatures, a decrease intemperature can be performed in combination with a reduction in feed.

Protein samples can be obtained from the cell cultures using methodsknown to those of skill in the art. For example, methods such asfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents can be used to disrupt cells to obtain protein samples.Where proteins are secreted by cells in the cell culture, a proteinsample can easily be recovered from the supernatant. Protein samples canalso be obtained using methods such as spheroplast preparation andlysis, cell disruption using glass beads, and cell disruption usingliquid nitrogen, for example.

Protein samples can also be further processed or purified before thecysteine column wash. For example, samples can be subjected to ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitcchromatography and lectin chromatography, protein refolding steps and/orhigh performance liquid chromatography (HPLC) either before or after thecysteine column wash.

VI. Compositions Comprising Proteins with Reduced Trisulfides and UsesThereof

Proteins that have been treated according to the methods describedherein to prevent and/or eliminate trisulfide bonds can be prepared forsubsequent use in diagnostic assays, immunoassays and/or pharmaceuticalcompositions.

In some embodiments, the protein, e.g. antibody, treated with themethods described herein has increased storage stability compared to anuntreated control. In another embodiment, the protein, e.g. antibody,treated with the methods described herein has a decreased tendency toaggregate compared to an untreated control. In still other embodiments,treatment of a protein, e.g. antibody, with the methods described hereinresults in decreased oxidation, e.g. methionine oxidation, as comparedto an untreated control.

Therefore, one embodiment provides a method for reducing proteinoxidation, e.g. methionine oxidation, in a composition of proteins, e.g.antibodies, comprising reducing the level of trisulfides in thecomposition of proteins. Another embodiment provides a method forreducing protein aggregation in a composition of proteins, e.g.antibodies, comprising reducing the level of trisulfides in thecomposition of proteins. Another embodiment provides a method ofincreasing protein stability in a composition of proteins, e.g.antibodies, comprising reducing the level of trisulfides in thecomposition of proteins.

In some embodiments, methods described herein can be used to increase orenhance long-term protein and antibody storage stability. Therefore, oneembodiment provides a method for improving long-term antibody storagestability by converting trisulfide bonds to disulfide bonds in anantibody wherein the method comprises: (a) allowing antibodies in asolution comprising at least one antibody with at least one trisulfidebond to contact and associate with a solid support; (b) exposing saidantibodies to a solution comprising a reducing agent; (c) replacing thesolution comprising the reducing agent with a solution lacking thereducing agent, and (d) preparing said antibodies in a composition forlong-term storage, wherein steps (a) and (b) are performedsimultaneously or in any sequential order, followed subsequently by step(c) then step (d). In one embodiment, improving long-term antibodystorage stability reduces cleavage or loss of covalent bonding ofantibody light chains from intact antibody molecules. In anotherembodiment, improving long-term antibody storage stability reduces theformation of antibody aggregates.

In some embodiments, the method of improving long-term antibody storagestability results in an improvement in long-term storage stability in anamount selected from the group consisting of: (a) at least about 5%improvement; (b) improvement of 5% or more; (c) at least about 10%improvement; (d) improvement of 10% or more; (e) at least about 15%improvement; (f) improvement of 15% or more; (g) at least about 20%improvement; (h) improvement of 20% or more; (i) at least about 25%improvement; (j) improvement of 25% or more; (k) at least about 30%improvement; (l) improvement of 30% or more; (m) at least about 40%improvement; (n) improvement of 40% or more; (o) at least about 50%improvement; (p) improvement of 50% or more; (q) at least about 60%improvement; (r) improvement of 60% or more; (s) at least about 80%improvement; and (t) improvement of 80% or more. The amount ofimprovement can be determined by comparing the antibody specific bindingactivity, percentage of intact antibody molecules, or percentage ofantibody aggregates in an antibody composition prepared according to themethods described versus an antibody composition not prepared accordingto the methods described. The improvement can be determined at any timeduring or after long-term storage.

In some embodiments, the long-term storage comprises storage for aperiod selected from the group consisting of: (a) 1 month or longer; (b)2 months or longer; (c) 3 months or longer; (d) 4 months or longer; (a)5 months or longer; (e) 6 months or longer; (f) 8 months or longer; (g)10 months or longer; (h) 12 months or longer; (i) 18 months or longer;(j) 24 months or longer; (k) 30 months or longer; (1) 36 months orlonger; (m) 48 months or longer; and (n) 72 months or longer. In someembodiments, the antibodies are stored in a substantially hydrated or asubstantially non-hydrated form.

In some embodiments, the long term storage comprises storage for aperiod of 1 month or longer at a temperature selected from the groupconsisting of: (a) −70° C. or below; (b) about −70° C.; (c) −70° C. orabove; (d) −20° C. or below; (e) about −20° C.; (f) −20° C. or above;(g) 0° C. or below; (h) about 0° C.; (i) 0° C. or above; (j) 4° C. orbelow; (k) about 4° C.; (l) 4° C. or above; and (m) 20° C. or below.

In some embodiments, the protein treated with the methods describedherein is formulated into a “pharmaceutically acceptable” form.“Pharmaceutically acceptable” refers to a bioproduct that is, within thescope of sound medical judgment, suitable for contact with the tissuesof human beings and animals without excessive toxicity or othercomplications commensurate with a reasonable benefit/risk ratio.

Antibodies treated according to the methods described herein can beformulated into pharmaceutical compositions for administration tomammals, including humans. The pharmaceutical compositions used in themethods of this invention comprise pharmaceutically acceptable carriers,including, e.g., ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

The compositions used in the methods of the present invention can beadministered by any suitable method, e.g., parenterally,intraventricularly, orally, by inhalation spray, topically, rectally,nasally, buccally, vaginally or via an implanted reservoir. The term“parenteral” as used herein includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional and intracranial injection orinfusion techniques.

A specific dosage and treatment regimen for any particular patient willdepend upon a variety of factors, including the particular antibodyused, the patient's age, body weight, general health, sex, and diet, andthe time of administration, rate of excretion, drug combination, and theseverity of the particular disease being treated. Judgment of suchfactors by medical caregivers is within the ordinary skill in the art.The amount will also depend on the individual patient to be treated, theroute of administration, the type of formulation, the characteristics ofthe compound used, the severity of the disease, and the desired effect.The amount used can be determined by pharmacological and pharmacokincticprinciples well known in the art.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed., Cold SpringHarbor Laboratory Press: (1989); Molecular Cloning: A Laboratory Manual,Sambrook et al., ed., Cold Springs Harbor Laboratory, New York (1992),DNA Cloning, D. N. Glover ed., Volumes I and II (1985); OligonucleotideSynthesis, M. J. Gait ed., (1984); Mullis et al. U.S. Pat. No.4,683,195; Nucleic Acid Hybridization, B. D. Hames & S. J. Higgins eds.(1984); Transcription And Translation, B. D. Hames & S. J. Higgins eds.(1984); Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc.,(1987); Immobilized Cells And Enzymes, IRL Press, (1986); B. Perbal, APractical Guide To Molecular Cloning (1984); the treatise, Methods InEnzymology, Academic Press, Inc., N.Y.; Gene Transfer Vectors ForMammalian Cells, J. H. Miller and M. P. Calos eds., Cold Spring HarborLaboratory (1987); Methods In Enzymology, Vols. 154 and 155 (Wu et al.eds.); Immunochemical Methods In Cell And Molecular Biology, Mayer andWalker, eds., Academic Press, London (1987); Handbook Of ExperimentalImmunology, Volumes I-IV, D. M. Weir and C. C. Blackwell, eds., (1986);Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989).

General principles of antibody engineering are set forth in AntibodyEngineering, 2nd edition, C. A. K. Borrebaeck, Ed., Oxford Univ. Press(1995). General principles of protein engineering are set forth inProtein Engineering, A Practical Approach, Rickwood, D., et al., Eds.,IRL Press at Oxford Univ. Press, Oxford, Eng. (1995). General principlesof antibodies and antibody-hapten binding are set forth in: Nisonoff,A., Molecular Immunology, 2nd ed., Sinauer Associates, Sunderland, Mass.(1984); and Steward, M. W., Antibodies, Their Structure and Function,Chapman and Hall, New York, N.Y. (1984). Additionally, standard methodsin immunology known in the art and not specifically described aregenerally followed as in Current Protocols in Immunology, John Wiley &Sons, New York; Stites et al. (eds), Basic and Clinical—Immunology (8thed.), Appleton & Lange, Norwalk, Conn. (1994) and Mishell and Shiigi(eds), Selected Methods in Cellular Immunology, W.H. Freeman and Co.,New York (1980).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein, J., Immunology: The Science of Self-Nonself Discrimination, JohnWiley & Sons, New York (1982); Kennett, R., et al., eds., MonoclonalAntibodies, Hybridoma: A New Dimension in Biological Analyses, PlenumPress, New York (1980); Campbell, A., “Monoclonal Antibody Technology”in Burden, R., et al., eds., Laboratory Techniques in Biochemistry andMolecular Biology, Vol. 13, Elsevere, Amsterdam (1984), Kuby Immunology4^(th) ed. Ed. Richard A. Goldsby, Thomas J. Kindt and Barbara A.Osborne, H. Freemand & Co. (2000); Roitt, I., Brostoff, J. and Male D.,Immunology 6th ed. London: Mosby (2001); Abbas A., Abul, A. andLichtman, A., Cellular and Molecular Immunology Ed. 5, Elsevier HealthSciences Division (2005); Kontermann and Dubel, Antibody Engineering,Springer Verlan (2001); Sambrook and Russell, Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Press (2001); Lewin, Genes VIII,Prentice Hall (2003); Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Press (1988); Dieffenbach and Dveksler, PCR PrimerCold Spring Harbor Press (2003).

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entireties.

EXAMPLES Example 1 Recombinant Antibodies Contain Trisulfide Bonds

An IgG1 monoclonal antibody (IgG1 mAb-A) expressed in Chinese HamsterOvary (CHO) cell clarified conditioned medium (CCM) was found to containa trisulfide modification via multiple analytical techniques. Massspectrum analysis showed a peak width broadening for the intact,non-reduced mAb. The average mass was increased, and both the massincrease and the peak broadening effect were eliminated by reducingconditions.

In these experiments, preparations of IgG1 mAb were first analyzed formonomeric, high and low molecular weight species by size exclusion highpressure liquid chromatography (SEC-HPLC) under isocratic conditions.The analysis was performed using a Tosoh Biosciences G3000SWXLanalytical column with a TSKgel guard column (#08543), a Waters 600Scontroller and a 717plus autosampler controlled by Empower software.Subsequent peptide mapping analysis was performed by high pressureliquid chromatography and mass spectrometry (LC-MS). Monoclonalantibodies (approximately 100 μg) were treated with 4-vinylpyridine atambient temperature for 30 minutes in the presence of approximately 6 Mguanidine hydrochloride. The protein was then recovered by ethanolprecipitation. Approximately 20 μg of the 4-vinylpyridine-treated mAbwas digested with endo-Lys-C (Wako, #125-02543) in the presence of 2 Murea at ambient temperature for 16 hours. An aliquot (approximately 4μg) was adjusted to 5 M urea and separated on a T3 HSS column in agradient of acetonitrile and trichloroacetic acid using a UPLC-LCTPremier system (Waters). Data generated by the liquidchromatography/mass spectrometry (LC-MS) analysis were processed usingMassLynx 4.1 software. The percentage of trisulfide-linked H-L peptideswas calculated from peak areas on the UV trace or on theextracted-ion-chromatogram of all three charged states, 1+ to 3+, withknown peptides used as internal standards. The percent H-L trisulfidewas calculated as (peak area of trisulfide)/(peak area of disulfide+peakarea of trisulfide). The limit of detection for the assay was <0.1%. Thelimit of quantification is <1%. These experiments confirmed the presenceof trisulfide bonds, and revealed that the predominant site ofmodification was the bond between the heavy and light chains (H-L bond).Modification of hinge region disulfides occurred at a lower frequency.In contrast, no trisulfide modification of intrachain disulfide bondswas detected.

In addition, it was observed that the IgG1 mAb-A preparation containingH-L trisulfide displayed increased sensitivity to extreme heat treatmentcompared to a similar preparation, termed “mAb-A-LT”, in whichtrisulfides were undetectable. In these experiments, analysis of IgG1mAb was performed by denaturing, non-reducing capillary electrophoresisin the presence of sodium dodecyl sulfate using an IgGPurity/Heterogeneity Assay Kit (# A10663) and ProteomeLab PA-800instrument (Beckman Coulter). Samples (200 μg) were prepared by heatingto 70° C. for 3 minutes in the presence of iodoacetamide, 1 mM SDS and a10 kDa standard essentially as per manufacturer's instructions (ProtocolA10424-AC) and separated for 35 min in a bare fused silica capillarywith detection at 220 nm. The results, shown in FIGS. 1A and 1B show anincreased level of fragmentation following sample preparation fornon-reducing electrophoresis. The electrophoretic migration pattern ofthe single most abundant fragment was consistent with the loss of onelight chain (L) from the intact mAb with the simultaneous generation ofa species consisting of two heavy chains and one light chain (HHLfragment). This phenomenon was then used to rapidly screen forconditions that influence trisulfide content in IgG1 mAbs. In thisscreen, non-reducing electrophoresis was used as a rapid assay fordetection of trisulfide bonds.

Example 2 Fermentation Conditions Affect Generation of Trisulfides

In order to determine if fermentation conditions affect trisulfide bondlevels, a series of studies was performed in bioreactors. An experimentwas performed to test the impact of fermentation time in a 200 literbioreactor using the anti-LINGO-1 antibody Li81. The time courserevealed a bell shaped curve. At early time points, trisulfide levelswere high. Trisulfide levels dropped as cultures achieved maximum celldensity and maximum antibody production, and the levels increased againat later time points. Based on these studies, day 13 was selected asideal for production.

Similar studies were performed in bioreactors of varying sizes: smallbioreactors (e.g., 3-6 liters), 200 liter bioreactors, and 2000 literbioreactors. The data suggest that it is possible to achieve lowertrisulfide bond levels via production of proteins in large volume cellcultures, for example, as cultured in large bioreactors.

Example 3 Treatment with Hydrogen Sulfide Increases Trisulfide Bonds

In order to determine if hydrogen sulfide concentrations in cell culturecould affect trisulfide bond levels, mAb-A containing 2% trisulfide wasexposed to increasing concentrations of hydrogen sulfide for increasingamounts of time. In these experiments, Mab (7.5 mg/mL) in 50 mM Tris pH7.8 was treated with 2.5, 0.5 or 0.125 mM H₂S diluted from a 125 mM H₂Swater solution (Ricca Chemical Company). Samples were incubated for 3hours RT, 24 hours RT, or 90 min at 37° C. and immediately desalted onP6DG spin columns equilibrated in 10 mM sodium citrate pH 6.5, 50 mMNaCl to remove excess H₂S. Samples were analyzed by peptide mapping. Theresults, shown in FIG. 2, demonstrate that both increases in either theconcentration of hydrogen sulfide or the time of exposure to hydrogensulfide correlated with increased trisulfide bonds.

Example 4 Cysteine Concentrations Affect Trisulfide Bonds

In order to determine if concentrations of cysteine affect trisulfidebond formation, trisulfide bond levels in cell cultures fed additionalcysteine were evaluated. On day 14, cultures were either fed a completefeed+supplemental amino acids+supplemental Cys; completefeed+supplemental Met+Cys; or complete feed+supplemental Met only. Theresults, shown in FIG. 3, demonstrate that increased trisulfide bondlevels in protein samples correlates with increased concentrations ofcysteine in a cell culture, at least when supplementation is performedlate in a cell culture run (e.g., at or after day 14).

Example 5 Characterization of Trisulfide Modification in Antibodies

Monoclonal antibodies (mAbs) are an important class ofbiopharmaceuticals for treatment of disease (Nieri et al., Curr. Med.Chem. 16 (2009) 753-779 and Walsh, Nat. Biotechnol. 24 (2006) 769-776).All share similar structural characteristics, high specificity, and longhalf-life. Successful development of biopharmaceuticals relies oncontinuous improvements in methods used to assess product heterogeneity.Common sources of heterogeneity are post-translational modifications,resulting from glycosylation, oxidation, deamidation, disulfide linkagescrambling, proteolytic cleavage, misfolding, etc. (Liu et al., J.Pharm. Sci. 97 (2008) 2426-2447). Some modifications such as oxidation,deamidation, and glycation occur during protein production, processing,and storage and can be detrimental (Jenkins et al., Mol. Biotechnol. 39(2008) 113-118), while others—glycosylation, phosphorylation, disulfidelinkage formation—are required for correct protein structure andfunction (Walsh, Posttranslational Modification of Proteins: ExpandingNature's Inventory, Roberts and Co. Publishers, 2005). Therefore,post-translational modifications are closely monitored to ensure theconsistency in product quality (Jenkins et al., Mol. Biotechnol. 39(2008) 113-118; Barnes and Lim, Mass Spectr. Rev. 26 (2007) 370-388; andZhang et al., Mass Spectr. Rev. 28 (2009) 147-176).

Antibodies are multi-domain proteins composed of two copies of light andtwo copies of heavy chains with multiple interchain and intrachaindisulfide bonds. Incorrect pairing of disulfide bonds may affect thefolding that can lead to a change in protein function, such as antigenrecognition, binding affinity, structure, and stability (Glockshuber etal., Biochemistry 31 (1992) 1270-1279). Commonly seen modifications indisulfide structure include disulfide bond scrambling (Glocker et al.,J. Am. Soc. Mass Spectr. 6 (1995) 638-643), glutathionylation (Galloglyand Micyal, Curr. Opin. Pharmacol. 7 (2007) 381-391), cysteinylation(Banks et al., J. Pharm. Sci. 97 (2008) 775-790), and oxidation.Trisulfide linkages in proteins, resulting from insertion of a sulfuratom into a disulfide bond, have been rarely documented. The presence ofa trisulfide in a protein was first reported for the minor disulfideloop of E. coli-derived recombinant human growth hormone (hGH)(Jespersen et al., Eur. J. Biochem. 219 (1994) 365-373; and Andersson etal., Int. J. Pept. Protein Res. 47 (1996) 311-321); later it was alsoseen in the major disulfide loop in a methionyl hGH (Canova-Davis etal., Anal. Chem. 68 (1996) 4044-4051). Trisulfides have also beendetected in a recombinant, truncated interleukin-6 expressed in E. coli(Breton et al., J. Chromatogr. A 709 (1995) 135-146.), and inCu,Zn-superoxide dismutase isolated from human erythrocytes(Okado-Matsumoto et al., Free Radic. Biol. Med. 41 (2006) 1837-1846).The trisulfide modification in hGH had no effect on function in vitro oron the pharmacokinetics of the compound (Thomsen et al., Pharmacol.Toxicol. 74 (1994) 351-358), but it did affect the hydrophobicity of theprotein. Methods including buffer exchange, pH control, and mildreduction have been developed for hGH to reduce trisulfide levels or toconvert the trisulfide back to a disulfide (U.S. Pat. No. 7,232,894).Recently, trisulfides were reported in the Cys linkages between theheavy chains of a recombinant human IgG2 mAb (Pristatsky et al., Anal.Chem. 81 (2009) 6148-6155). We report here the discovery of trisulfidelinkages between the light and heavy chains in preparations ofrecombinant and natural IgG 1, 2, 3, and 4 isotypcs. The amount oftrisulfide in mAbs varies and occurs predominately in the linkagebetween the light and heavy chains. A mass spectrometric method fortheir determination and quantification, the impact of fermentationconditions on trisulfide formation, and the effect of trisulfidemodification on antibody activity and stability is al so discussed.

Materials and Methods

Monoclonal antibodies [mAb1 (aglycosyl form), mAb2 (wild type andaglycosyl forms), mAb3, mAb4, mAb5, mAb6, and mAb7] were produced in CHOcells and purified by protein A affinity chromatography at Biogen Idec(Cambridge, Mass.). Natural human myeloma IgGs were obtained fromSigma-Aldrich (St. Louis, Mo.). Product numbers were I 5154 for IgG1, I5404 for IgG2, I 5654 for IgG3, and I 4639 for IgG4. Commercial mAbpharmaceuticals (referred to as Drug 1, Drug 2, and Drug 3) wereobtained through appropriate agencies.

Intact Mass Analysis. About 22.5 μg of the native protein was analyzedon an LCT electrospray time-of-flight mass spectrometer (Waters,Milford, Mass.). Prior to analysis the protein was desalted using anarrowbore Vydac C4 cartridge (5 μm particle size, 2.1 mm I.D.)connected to an Acquity UPLC system (Waters, Milford, Mass.). Mobilephase A was 0.03% trifluoroacetic acid in water, and mobile phase B was0.024% trifluoroacetic acid in acetonitrile. The proteins were desaltedusing 100% A for 5 min, then eluted with 80% B in 1 min at flow rate of0.1 mL/min. Molecular masses were obtained by deconvolution using theMaxEnt 1 program. Average peak widths in the raw mass spectra weredetermined from charge states 49 to 51 by measuring the full width athalf maximum (FWHM).

Alkylation of Proteins. About 1.0 μL of a 1:10 dilution of4-vinylpyridine (in 8 M guanidine hydrochloride) was added to 12.5 μL ofa solution containing ˜90 μg of the protein, and 8 M guanidinehydrochloride was added to the mixture to give a final volume of 50 μL.The solution was kept at 20° C. in the dark for 30 min. The alkylatedprotein was recovered by precipitation with 40 volumes of cooledethanol. The mixture was stored at −20° C. for 1 h and then centrifugedat 14000 g for 12 min at 4° C. The supernatant was discarded and theprecipitate (˜22.5 μg/vial) was washed once with cooled ethanol.

Non-reduced Tryptic and Endoprotease Lys-C Peptide Mapping. For trypticdigestion, about 22.5 μg of the 4-vinylpyridine-treated protein wasdigested with 5% (w/w) of endo-Lys-C (Wako Chemical, Richmond, Va.) in50 μL of 2 M urea, 0.2 M Tris.HCl, 2 mM CaCl₂, pH 6.5, for 5 h at roomtemperature, after which 10% (w/w) of trypsin (Promega, Madison, Wis.)was added and the solution was kept at room temperature for additional15 h. Prior to analysis of the digests by LC-MS, 50 μL of 8 M urea wasadded to each digest, and the digest was split equally into two vials:the sample in one vial was reduced with of 30 mM TCEP in 150 mM Tris, pH6.0, 1 mM EDTA, at 37° C. for 1 h; the other portion was analyzeddirectly without the reduction. Both the reduced and non-reduced digestswere analyzed on a Waters UPLC-LCT Premier XE system (Waters, Milford,Mass.). Mobile phase A was 0.03% trifluoroacetic acid in water, andmobile phase B was 0.024% trifluoroacetic acid in acetonitrile. Analiquot of 50 pmole of the digests was separated on a 2.1×150 mm T3 HSScolumn with a gradient of 0% to 40% B over 60 min. The flow rate was 70μL/min.

For focused Lys-C peptide mapping, about 22.5 μg of the4-vinylpyridine-treated protein was digested in 50 μL of 2 M urea, 2 mMCaCl₂, 0.2 M Tris-HCl (pH 6.5), with 10% (w/w) of Lys-C (Wako) for 16 hat room temperature. Prior to analysis of the digests by LC-MS, 50 μL of8 M urea was added to each digest. An aliquot of the digest of 25 pmolnative protein (3.8 μg) was analyzed on the same LC-MS system, exceptthat the gradient was from 10% to 19.5% B in 10 min. The percentages ofthe trisulfide-linked peptides were calculated from peak areas on eitherthe UV trace or extracted ion chromatogram (EIC).

Spiking experiments. Samples of both mAb2-agly C (which contains 7.8%trisulfide at the LC5-HC5 linkage) and mAb2-agly D (which has nodetectable trisulfide) were diluted to 1.0 mg/mL with PBS. Theconcentration of the protein was calculated from its UV absorbance at280 nm using a calculated extinction coefficient [A₂₈₀ (1 mg/mL)=1.44mL/mg cm]. To determine the detection limit of the trisulfide at theLC5-HC5 linkage, different amounts (0.56%-50%) of mAb2-agly C werespiked into mAb2-agly D, using low-volume microsyringes. An aliquotcontaining 25 pmole (3.8 μg) of the digest was injected for Lys-Cpeptide mapping analysis with the lowest concentration of trisulfidespike being run first, followed by increasingly higher amounts of spike.The samples were analyzed in duplicate in each case. Between sampleruns, the column was cleaned by injecting water and running thegradient. The amount of the trisulfide was estimated from peak areas inextracted ion chromatograms (EIC) for the disulfide-linked and thecorresponding trisulfide-linked peptide clusters containing the LC5-HC5linkage.

Incubation with H₂S. The protein (mAb1, 7.5 mg/mL) in 50 mM Tris buffer(pH 7.8) was treated with 0.5-, 2.0- or 2.5-mM H₂S diluted from a 125-mMH₂S water solution (Ricca Chemical Co., Arlington, Tex.). Samples wereincubated for 3 h or 24 h at room temperature, or 90 min at 37° C. Afterincubation, samples were immediately desalted on Bio-gel P6DG spincolumns (Bio-Rad, Carlsbad, Calif.) equilibrated in 10-mM sodium citrate50 mM NaCl, pH 6.5, in order to remove excess H₂S. Samples were analyzedby peptide mapping.

Analysis of mAb 1 and mAb2 Functions by Direct Binding ELISA. Costar96-well easy wash plates were coated overnight with 5 μg/mL humanantigen that binds to mAb1 (50 μL/well), in 50 mM sodium carbonate (pH9.5) at 4° C. Plates were washed 3 times with 350 μL of PBS using aplate washer and blocked with 1% BSA, 0.1% ovalbumin, and 0.1% non-fatdry milk in Hank's balanced salt buffer containing 25 mM HEPES (pH 7.0)for 1 hour at room temperature. Plates were then washed again, andincubated for 1 h with a 3-fold dilution series set up for each testcompound (50 μL/well, 8 dilutions) starting at 10 μg/mL and all in 0.1%BSA, 0.1% ovalbumin, and 0.1% non-fat dry milk in Hank's balanced saltbuffer containing 25 mM HEPES (pH 7.0), and again washed. Bound mAb1 wasdetected using AP-anti-human Fab. Plates were incubated for 1 h at roomtemperature, washed, and treated with 10 mg/mL 4-nitrophenylphosphatealkaline phosphate substrate in 100 mM glycine, pH 10.5, 1 mM MgCl₂, 1mM ZnCl₂. Plates were read at 405 nm on a Molecular Devices platereader. EC₅₀ values for binding were calculated from the titration curveusing prism software. The function of mAb2 was assessed as for mAb1except that 96 well microplates were coated overnight at 4° C. with 1μg/mL (in 15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6) of a human antigen thatbinds to mAb2. Plates were blocked for 1 h at room temperature with PBScontaining 2% BSA. Antibodies were diluted to the indicatedconcentrations and incubated for 2 h at room temperature. Binding wasdetermined using a horseradish peroxidase-conjugated goat anti-humanpolyclonal antibody (Jackson ImmunoResearch, West Grove, Pa.) followedby measurement of horse radish peroxidase activity using the substratetetramethylbenzidine.

Assessing the Stability of Trisulfides in Rat Serum in vitro and invivo. For in vitro studies, 500 μg of mAb 1-B (20% trisulfide in thelight-heavy chain linkage) was incubated in PBS or in 1 mL of normal ratserum at 37° C. for 16 or 96 h, after which time the protein waspurified on IgSelect affinity resin (GE Healthcare, Piscataway, N.J.).The samples were incubated with 100 μL of the resin for 2 h at roomtemperature with mild agitation. The resin was then washed six timeswith 0.5 mL of PBS and six times with 0.5 mL of 25 mM sodium phosphate,100 mM NaCl, pH 5.5, after which the bound antibody was eluted with 25mM sodium phosphate, pH 2.8, 100-mM NaCl. The eluate was neutralized bythe addition of 1/20 volume of 0.5 M sodium phosphate, pH 8.6. For invivo studies, three 200-g Sprague Dawley rats were injectedintraperitoneally with 100 mg/kg mAB1-B. The blood was drawn after 24and 72 h, and allowed to clot to form serum. Portions of the serum (750μL for the 24-h point, and 1050 μL for the 72-h point) were subjected toIgSelect purification using 150 μL of IgSelect (protocol describedabove). The purified mAb1 was analyzed by non-reduced SDS-PAGE and Lys-Cpeptide mapping to determine the trisulfide level. The reisolated mAb1samples from the in vitro and in vivo studies were intact and >95% pureby SDS-PAGE. Based on column recoveries, levels of mAb1 in the serumafter 24 and 72 h were 850 and 700 μg/mL of mAb1, respectively, whichmatched the theoretical predictions generated from rat pharmacokineticstudies of mAb1 samples lacking trisulfide.

Results

Intact Mass Analysis. The presence of trisulfides in monoclonal antibody1 (mAb1) was anticipated from intact mass measurements where an increaseof up to 20 Da was observed in some non-reduced samples, but not inreduced ones. The increase in mass was accompanied by a broader thanexpected peak width in the raw mass spectrum indicating productheterogeneity. The magnitude of the increase in mass and peak broadeningdirectly correlated with the level of trisulfide, which was confirmed bypeptide mapping. The mass spectrum of mAb1-B (an aglycosyl IgG1antibody), which has 20% trisulfide at the LC5-HC5 linkage was overlaidon the mass spectrum of mAb1-A, which as 0.5% trisulfidc at the LC5-HC5linkage, at charge +50. Peak broadening resulted in a shift in theobserved mass of the protein from 144,440 Da to 144,459 Da.

Peptide Mapping of non-reduced proteins and Tandem Mass spectrometry.Fully characterized peptide maps of mAb 1 were developed foridentification and quantification of the trisulfide modification.Tryptic peptide mapping of reduced mAb1 (reduced map) confirmed theamino acid sequences, but did not reveal any modification that couldaccount for the mass increase observed by intact mass measurement ofnative mAb1-B. Peptide maps of non-reduced protein were used to studythe peptide linkages that form the disulfide structure of the protein.As shown schematically in FIG. 4A, IgG1 antibodies contain 12 intrachainand 4 interchain disulfides. Tryptic peptide mapping of non-reducedmAb1-B revealed all the predicted disulfide-linked peptide clusters asmajor components in the peptide map. Three extra peaks, eluting at 16,19, and 54 min in the tryptic map of the non-reduced protein that werenot present in the peptide map of the reduced protein were alsodetected. The molecular masses of the components in these peaks are788.217, 1292.457, and 5486.767 Da, respectively. Extracted ionchromatograms (EIC) of the peptides showed eluting at 16 and 19 min,respectively. The mass of 788.217-Da component is 32 Da higher than thecalculated mass (756.242 Da) for the disulfide-linked peptide clusterPepLT20/PepHT17 [i.e., the tryptic peptide #20 of the light chain(PepLT20) and tryptic peptide #17 of the heavy chain (PepHT17) arelinked through Cys residues]. Similarly, the mass of the 1292.457-Dacomponent is 32 Da higher than the mass calculated for thedisulfide-linked peptide cluster PepLT19-20/PepHT17 (1260.486 Da), whicharises by partial digestion of the protein (i.e., the peptide bondbetween peptides PepLT19 and PepLT20 has not been cleaved). The peptideclusters that elute at 16 and 19 min both contain Cys residue 5 of thelight chain (LC5) and Cys residue 5 of the heavy chain (HC5) which formthe interchain disulfide that links the heavy chain and light chain(FIG. 4).

Tandem mass spectrometry was performed on these peptides to confirm thetype and location of the modification. The CID mass spectra obtainedfrom the doubly charged ions of the disulfide-linked and the presumedtrisulfide-linked PepLT19-20/PepHT17 peptide clusters were analyzed.Fragment ions lacking a Cys residue showed the same masses in bothspectra. However, all the fragment ions containing Cys showed 32-Dadifferences in the corresponding CID spectrum. For example, m/z of they1 ion is 571.25 for PepLT19-20/PepHT17 disulfide cluster, but 603.08for the corresponding trisulfide cluster. Furthermore, some of thedisulfide and trisulfide bonds were cleaved during the CID experiment.In the CID spectrum of the trisulfide-linked peptide cluster,PepLT19-20+2S (876.33 Da) and PepHT17+2S (516.17 Da) were clearlyobserved while these ions were absent in the CID spectrum of thecorresponding disulfide-linked peptide cluster.

The peak eluting at 54 min in the peptide map of the non-reduced proteinwas subjected to the same analysis. The 5486.767-Da component is 32 Dahigher than the mass calculated for the disulfide-linked hinge peptidesof the heavy chain (PepHT18-PepHT18, 5454.783 Da), in which HC6 and HC7on one heavy chain are linked to the same residues on the other heavychain. Both pairs of Cys residues must be involved in a trisulfidelinkage because there were two overlapping peaks in the EIC for thetrisulfide-linked cluster of the heavy chain (data not shown). However,the exact linkage could not be determined by CID MS/MS because of thelow abundance of these species.

The EIC data for the modified and unmodified LC5-HC5 and HC6/7-HC6/7peptide clusters were used to quantify trisulfide levels in the mAb1-Bsample. Trisulifide bonds in mAb 1-B accounted for 20% of the LC5-HC5linkage and 4% of the HC6/7-HC6/7 linkages.

Trisulfides in Other Antibodies. Natural and recombinant antibodypreparations representing all IgG subclasses were examined in order tobetter understand the frequency of occurrence of trisulfide linkages inantibodies. Tables 12 and 13 show all subclasses of IgG antibodiescontain trisulfides.

Table 12 summarizes results generated with 7 non-clinical recombinantmonoclonal antibodies. Trisulfide levels at the LC5-HC5 linkage in themAb1 preparations ranged from 0.2-39.3%. Trisulfide levels at theLC5-HC5 linkage of the mAb2 preparations (wild type and aglycosyl IgG1)varied from <0.1% to 21%. Trisulfide levels in the HC6/7-HC6/7 linkageswere considerably lower than in the LC5-HC5 linkage and demonstratedthat the LC5-HC5 connection was the most sensitive site for trisulfidemodification. No trisulfides were detected in intrachain disulfidelinkages. In IgG2, IgG3, and IgG4 mAbs, the heavy and light chains arejoined via a LC5-HC3 disulfide bond in place of the LC5-HC5 linkagefound in IgG1 mAbs. Trisulfide levels in the LC5-HC3 linkage of two IgG4mAbs, mAb5 and mAb6, were 11% and 5%, respectively. Trisulfide levels inthe HC6/7-HC6/7 linkages were 4% for mAb5 and <1% for mAb6 (Table 12 andFIG. 4D) and no intrachain trisulfides were detected. Trisulfide levelsat the LC5-HC3 linkage of IgG 2 mAb3 ranged from 0.4 to 6% (Table 12 andFIG. 4B), and there was no detectable trisulfide in the hinge andintrachain disulfide linkages. Trisulfide levels at the LC5-HC3 linkageof IgG3 mAb4 was 5% (Table 12 and FIG. 4C). Monoclonal antibody 7(mAb7), a chimeric human/murine IgG2a antibody, contained 19-28%trisulfides at LC5-HC3 and 0-1% trisulfides in each of the three Cyslinkages between the heavy chains (Table 12).

TABLE 12 Trisulfide levels in recombinant antibodies detected using thedetailed peptide mapping method Trisulfide (%) Between Between Number ofthe light and the heavy Sample Preparations heavy chains chains Cyslinkages mAb1 7  0.2-39.3 0-6.8 LC5-HC5 (human IgG1) HC6/7-HC6/7 mAb2 3<0.1-15.8 0-4.7 (human IgG1) mAb3 11 0.4-6.3 Not LC5-HC3 (human IgG2)observed mAb4 1 11.0 Not LC5-HC3 (human IgG3) observed mAb5 1 10.8 4.0LC5-HC3 (human IgG4) HC6/7-HC6/7 mAb6 1  5.2 Not (human IgG4) observedmAb7 4 19.0-28.1 0-3.0 LC5-HC3 (murine IgG2a) HC6/7/8- HC6/7/8 mAb1 90 0.2-39.3 0-6.8 LC5-HC5 (human IgG1) HC6/7-HC6/7 agly mAb2 135 <0.1-20.60-4.7 (human IgG1) wt + agly mAb3 12  2.9-13.0 Not (human IgG1) observedmAb4 11 0.4-6  Not LC5-HC3 (human IgG2) observed mAb5 1 11.0 Not LC5-HC3(human IgG3) observed mAb6 1 10.8 4.0 LC5-HC3 (human IgG4) HC6/7-HC6/7mAb7 1  5.2 Not (human IgG4) observed mAb8 4 19.0-28.1 0-3.0 LC5-HC3(murine IgG2a) HC6/7/8- HC6/7/8

Table 13 summarizes peptide mapping results from additional studieswhere three commercial IgG1 therapeutic mAbs and natural preparations ofIgG1, IgG2, IgG3, and IgG4 samples isolated from human myeloma plasmawere characterized. The three commercial IgG1 therapeutic mAbs contained1-4% trisulfide at the LC5-HC5 linkage. The IgG1, IgG2, IgG3, and IgG4samples isolated from human myeloma plasma contained ˜1% trisulfide atthe linkage between the light and heavy chains (LC5-HC5 for IgG1 andLC5-HC3 for the rest; Table 13 and FIG. 4) and no other trisulfidelinkages were detected. These results demonstrate that trisulfides canoccur in any antibody, regardless of subclass, glycosylation state, orwhether it is an endogenous or recombinant protein.

TABLE 13 Trisulfide levels in commercial therapeutic IgG1 antibodies andin human myeloma IgG1, IgG2, IgG3, and IgG4 antibodies. Trisulflde inCys- linkage between the light and heavy chains Sample Notes (%) Cyslinkages Drug 1 (IgG1) 4.1 LC5-HC5 Drug 2 (IgG1) 0.5 Drug 3 (IgG1) Lots1-3 1.4-3.1 Human IgG1 from human 0.6 myeloma plasma Human IgG2 fromhuman 1.1 LC5-HC3 myeloma plasma Human IgG3 from human 1.5 myelomaplasma Human IgG4 from human 0.9 myeloma plasma

Focused Peptide Mapping for Monitoring Trisulfide Linkages in IgG1 mAbs.A focused Lys-C peptide mapping method that monitors the LC5-HC5 linkagewas developed to increase sample throughput. The LC5-HC5 linkage wastargeted because the level of the trisulfide linkage between the lightand heavy chains was consistently higher than that between the two heavychain for IgG1 mAbs. FIG. 5A shows the EICs (I and II) and UV trace(III) of the disulfide and trisulfide linked peptide clusters containingLC5 and HC5 in the Lys-C peptide map of the non-reduced protein. Thepercentage of trisulfide modified LC5-HC5 shown in FIG. 5A-III is about20%, the same as that seen in the tryptic peptide map. The focusedpeptide mapping method, utilizing the simplified digestion process and areduced separation time from 70 min to 15 min, was applied to allsubsequent trisulfide analyses of IgG1 antibodies. The trisulfidedetection limit for this method at the LC5-HC5 linkage, assessed byspiking experiments, is 0.1% (FIG. 5B; R²=0.9989), and standarddeviation of the method, based on more than twenty individualexperiments of a single preparation is 0.07 (relative standard deviationis 3.8%, FIG. 5C). More than two hundred preparations of IgG1 mAb1 andmAb2 were analyzed using the focused peptide mapping method in supportof studies designed to understand the impact of fermentation conditionsin bioreactors on trisulfide formation (see below).

Effect of Cell Culture Conditions on Trisulfide Formation. Evaluation oftrisulfide levels in mAb1 from 21 bioreactor runs revealed that cellculture conditions had a very dramatic effect on trisulfide formation.Time course studies from four 200-L bioreactors were run under nearidentical conditions, and the four bioreactors all showed a similarprofile where trisulfide levels were minimal around day 12 or day 13 andconsiderably higher earlier or later (e.g. at day 9 or day 16).Bioreactor studies with mAb2 (wild type and aglycosyl forms) showed asimilar trend. Trisulfide levels in mAb1 samples from 2000-L bioreactorsusing conditions developed at the 200 L scale showed similarconsistency. To investigate other variables that might impact trisulfideformation for mAb1, a number of cell culture parameters such as celldensity and feed strategies were varied in 3 to 6-L bioreactor studies.Trisulfide analysis of the samples from these runs revealed that thetrisulfide content in the LC5-HC5 linkage varied considerably from <1%to 39%, and the measurements from the 200 L and 200 L sizes were lessthan 5%. Although evaluation of culture conditions showed no simplecorrelation between the levels of trisulfides and any identifiableparameter, these studies indicate that cell culture conditionssignificantly affect trisulfide levels in mAbs.

Effect of H₂S Concentration on the Trisulfide Level. Studies withrecombinant hGH made in E. coli indicated that the trisulfide levelcorrelates with H₂S produced during fermentation (U.S. Pat. No.7,232,894). In an attempt to determine whether the H₂S concentrationaffects the level of the trisulfide in antibodies, a sample of mAb1(mAb1-A) with 0.5% trisulfide in LC5-HC5 linkage was incubated withdifferent concentrations of H₂S. Similar to the observations with hGH(U.S. Pat. No. 7,232,894), trisulfide formation in mAb1 is dependent onthe concentration of H₂S in the buffer (Table 14). Incubation with 0.5mM H₂S at 37° C. for 90 min increased the trisulfide level from 0.5% to5%. The highest trisulfide levels obtained in the presence of H₂S wereabout 15%. Prolonged incubation did not increase the levels. Detailedtryptic peptide mapping analysis of these samples showed that the sulfuraddition occurred only in the interchain Cys linkages, as was observedin the mAb1 preparations isolated from bioreactors.

TABLE 14 Effect of H₂S on trisulfide levels at the LC5-HC5 linkage.Trisulfide Sample Reagent Experimental Conditions (%) mAb1-A untreated /0.5 H₂S 0.5 mM, 37° C., 90 min 4.3 H₂S 2.5 mM, 37° C., 90 min 7.7 H₂S0.5 mM, Room Temperature, 3 h 4.5 H₂S 2.5 mM, Room Temperature, 3 h 15.4H₂S 0.5 mM, Room Temperature, 24 h 4.6 H₂S 2.5 mM, Room Temperature, 24h 14.7

Effect of Trisulfides on Antibody Binding to Antigen. The bindingaffinity of a mAb1 sample (mAb1-C) containing 39% trisulfide at theLC5-HC5 linkage was compared with that of mAb1-A containing about 0.5%of trisulfide at the LC5-HC5 linkage using an ELISA-based binding assay.Both of the samples produced the same sigmoidal binding curve withcalculated EC₅₀ values of 20 pM. Furthermore, an in vitro bioassayshowed that activities of both mAb1 samples were indistinguishable. Thebinding affinities of wild type and aglycosyl mAb2 with high and lowtrisulfide levels at the LC5-HC5 linkage were also compared. As alsoseen for mAb1, all mAb2 samples displayed essentially the same sigmoidalbinding curve with EC₅₀ values of about 35 pM. The similarity of thebinding curves suggests that the amount of trisulfide in the proteinsdid not affect the binding properties of mAb1 and mAb2.

In vitro and in vivo Stability of Trisulfides. The stability oftrisulfide in mAbs was assessed in a defined buffer system, in rat scrumin vitro and in vivo in rats following systemic administration. In thedefined buffer system experiment, mAb1-B (containing 20% trisulfide atthe LC5-HC5 linkage) and mAb1-D (containing 3% trisulfide at the LC5-HC5linkage) were incubated at 5° C. and at 25° C. in citrate buffer at pH6.5 for periods of one and three months. Peptide mapping analysisrevealed that the trisulfide linkages were stable and that there was nochange in the protein quality under these conditions. To assess thestability of the trisulfide in serum in vitro and in vivo, mAb1-Bcontaining 20% trisulfide was incubated in rat serum at 37° C. for 16 hand 96 h; the same material was also injected into rats and isolatedfrom the serum 24 and 72 h post injection. No change in the level of thetrisulfide was detected in the samples incubated in rat serum in vitrocompared with the untreated control (Table 15; FIG. 6). In contrast, notrisulfide was detected in the sample that had circulated in rat for 24h and for 72 h following intraperitoneal injection. Since the recoveryof the protein from the injected serum was essentially quantitative (seemethods section), the data suggest that complete conversion of thetrisulfide to disulfide occurs in vivo during the first 24 h ofcirculation. Furthermore, the levels of disulfides, observed in peptidemap, were the same as those in the map of a mAb1 containing littletrisulfide.

TABLE 15 Stability of trisulfides in rat serum in vitro and in vivo.Trisulfide at LC5-HC5 Description of Samples of mAb1-B (%) Control 19.8Control, purified by IgSelect 19.8 purified by IgSelect after incubatedat 37° C. 20.0 in rat serum for 16 h purified by IgSelect afterincubated at 37° C. 20.6 in rat serum for 96 h IgSelect purified aftercirculation in rat for 24 h <0.1 IgSelect purified after circulation inrat for 72 h <0.1Discussion

We have demonstrated that trisulfides are a common post-translationalmodification in antibodies and can occur in all IgG frameworks,independent of their glycosylation state. The modification can occur inrecombinant and natural antibodies as well as in non-clinicalpreparations and commercial therapeutics. Trisulfide levels in thesamples varied from below the detection limit to >40%, but the presenceof high levels of trisulfide had no observable effect on the function orstability of the antibody. Although there is very little publishedliterature on trisulfides in proteins, our findings suggest that it is avery common modification. The scarcity of published data presumablyarises because trisulfide detection requires the use of methods such asthose described here that are directly targeted at their analysis.

Peptide mapping of non-reduced protein samples utilizing a LC-MS systemis a sensitive and reliable method for identifying and quantifyingtrisulfides. In all IgG antibodies that were evaluated, trisulfidesoccurred in the heavy-light and heavy-heavy interchain linkages thatconnect the four subunits and not in any of the intrachain disulfides.The highest levels were consistently observed in the heavy-light chainlinkage. To increase the throughput and to simplify the peptide mappinganalysis, a focused Lys-C peptide mapping method for human IgG1 wasdeveloped. This protocol shortened analysis time significantly. Thepercentages of the trisulfide in the light and heavy chain linkage canbe easily quantified from chromatograms using either UV traces or EICs.Quantification from the EIC is preferred when the level of trisulfide isbelow 3%. The detection limit of the method estimated from EICs is 0.1%,with a RSD of 3.8% based on more than twenty independent experiments.Because the peptide sequences containing LC5 and HC5 are the same in allhuman IgG1 antibodies, the Lys-C peptide mapping method developed formAb1 is applicable to all human IgG1 antibodies. Similar peptide mappingstrategies were successfully developed for quantification of trisulfidelinkages in IgG2, IgG3, and IgG4 antibodies. Previously, Pristatsky andcoworkers (Pristatsky et al., Anal. Chem. 81 (2009) 6148-6155)identified trisulfide modification of the hinge of a human IgG2 mAb aspart of the characterization of the hinge disulfide structure. Tofacilitate characterization, the mAb had been fractionated on an ionexchange column. In the IgG2 mAb that we characterized—withoutfractionation—the primary site of modification was in the LC5-HC3linkage and non-detectable levels were observed in the hinge region.Because of the low level of trisulfide in LC5-LC3 linkage in ourpreparation, levels in the hinge may have been below the limit ofdetection.

The effect of cell culture conditions on the trisulfide levels wassurprising. In the nearly 100 mAb1 preparations that were purified andcharacterized, the level of trisulfide at the LC5-HC5 linkage rangedfrom <1% to 40%. Changes in culture conditions that seemed relativelyminor and did not significantly affect growth and culture productivityresulted in large differences in trisulfide levels. In particular,culture duration and feeding strategy were important variables andproduct with reproducible trisulfide levels can be obtained by tightlycontrolling cell culture conditions.

We have shown that trisulfides can be chemically incorporated intoantibodies by simple treatment with H₂S, confirming the original findingwith hGH (U.S. Pat. No. 7,232,894). Production of H₂S by mammalian cellsand tissues through the enzymatic breakdown of cysteine and homocysteineis well documented (Chiku et al., J. Biol. Chem. 284 (2009) 11601-11612;Kamoun, Amino Acids 26 (2004) 243-254; Singh et al., J. Biol. Chem. 284(2009) 22457-22466; Stipanuk and Beck, Biochem. J. 206 (1982) 267-277;and Zhao and Wang, Am. J. Physiol. Heart Circ. Physiol. 283 (2002)H474-480) and could account for trisulfide formation during cellfermentation. The specificity of the chemical reaction is driven bysolvent exposure of the interchain disulfides and by lack of exposure ofintrachain disulfides, which are buried in the hydrophobic interior ofantibodies. The low levels of H₂S in human tissue (Goodwin et al., J.Anal. Toxicol. 13 (1989) 105-109; and Zhao et al., Embo J. 20 (2001)6008-6016) could account of the trisulfide we observed in the endogenousmyeloma human IgG1, IgG2, IgG3, and IgG4 antibodies, and suggests thatpeople may be routinely exposed to proteins containing trisulfidelinkages.

The trisulfide linkage was stable to prolonged storage at 4° C. and atroom temperature and in rat serum in vitro, but was rapidly converted toa disulfide within 24 h after systemic administration to rats. The rapidconversion of the trisulfide to a disulfide in rats is consistent withother reported reduction-oxidation related changes that occur in vivo,e.g. the rearrangement of human IgG2 antibody hinge disulfides (Liu etal., J. Biol. Chem. 283 (2008) 29266-29272) and Fab-arm exchange ofhuman IgG4 (Labrijn et al., Nat. Biotechnol. 27 (2009) 767-771). All ofthese pathways require that an interchain linkage be reduced and thenreformed. In the absence of a reductant, the trisulfide linkage is verystable but reversal of the trisulfide to a disulfide can occur both invitro and in vivo by a mild reduction-oxidation process.

In summary, we have found that insertion of a sulfur atom into aninterchain disulfide bond to form a trisulfide is a commonpost-translational modification that has gone largely undetected becausetargeted methods are needed for their detection. The methods that weredeveloped for identification and quantification of trisulfides should beapplicable to any antibody and can be easily adapted for thecharacterization of other types of proteins. In the future, theidentification and quantification of trisulfides will undoubtedly becomean important test in the characterization of biopharmaceuticals.

Example 6 Full Peptide Mapping

To further analyze the effect of the on-column trisulfide conversionprocedure non-reducing peptide mapping experiments were performed.Specifically, non-reducing peptide mapping was performed for three mAb-AProtein A eluate samples corresponding to (1) untreated mAb-A-LT (1.3%trisulfide), (2) mAb-A (17% trisulfide) untreated, or (3) mAb-A (17%trisulfide) subjected to a 1 mM cysteine column-wash treatment. Thethree samples (approximately 100 μg) were denatured and alkylated for anhour at 25° C. in 115 μL of 8 M guanidine, 0.1 mM NEM in 50 mM sodiumphosphate buffer, pH 6.5. The samples were then diluted by mixing with246 μL of 50 mM sodium phosphate buffer, pH 6.5 before an addition of 10μg lysyl endopeptidase (Lys-C, Wako Chemicals). The digestion wasperformed at 25° C. for 18 hours. An aliquot of 90 μL of the proteindigest (equivalent to 25 μg of protein) was separated on a YMC ODS-Acolumn (2×250 mm, 5 μm, 120 Å) in a gradient of water, ACN with TFA,using an Agilent 1000 HPLC equipped with an ESI mass spectrometer (LCQDeca XP, Thermo). The peptide map data were explored for the presence oftrisulfide-modified, disulfide scrambled or free-cysteine containingpeptides. The percentage of trisulfide modification was calculated as(peak area of trisulfide peptide)/(peak area of disulfide peptide+peakarea of trisulfide peptide).

The level of H-L trisulfide in mAb-A was greatly decreased following theon-column 1 mM cysteine wash, from an initial level of 17% to apost-treatment level of 1.5%, which was similar to the level detected inuntreated mAb-A-LT (1.3%). Moreover, the disulfide bonded peptide mapfor each sample was similar, with no detectable differences in the levelof disulfide scrambling or free mAb sulfhydryl groups.

Example 7 Conversion of Trisulfides does not Affect Protein Structure orFolding

To assess the impact of trisulfide-to-disulfide conversion on proteinconformation, hydrogen/deuterium exchange mass spectrometry (H/DX-MS)was performed to measure deuterium incorporation into IgG1 mAb-A. Thistechnique has been used to explore structural changes and conformationaldynamics of IgG1 molecules. Houde et al. Anal. Chem. 81: 2644 (2009).The H/DX was performed as described in Houde et al. with a labeling timecourse of 10 sec, 1, 10, 60 and 240 min. Deuterated samples werequenched at pH 2.6 by the addition of an equal volume of H₂O containing200 mM sodium phosphate, 1 M TCEP and 4 M guanidine HCl, pH 2.3 withchilling to 0° C. Quenched samples were digested, desalted and separatedonline using a Waters UPLC based on a nanoACQUITY platform (Milford,Mass.) Wales et al., Anal. Chem 80 (2008) 6815). Approximately 20 pmolesof exchanged/quenched IgG1 was injected into the immobilized pepsincolumn. Digestion time was 2 min with a flow rate of 0.1 mL/min in 0.05%formic acid (FA) at 15° C. Peptic peptides were trapped on an ACQUITYUPLC BEH C18 1.7 μm peptide trap at 0° C. and desalted with water, 0.05%FA. Trapped peptides were eluted onto an ACQUITY UPLC BEH C18, 1.7 μm,1×100 mm column for separation at 40 μL/min and separated in a linearACN gradient (5-50%, 8 min) with 0.05% FA. Eluate was directed into aWaters Synapt HD MS with electrospray ionization and lock-masscorrection (using Glu-fibrinogen peptide). Mass spectra were acquiredfrom m/z 255 to 1800. Pepsin fragments were identified by a combinationof exact mass and MS/MS, aided by Waters IdentityE software. Silva etal. Mol. Cell. Proteomics 5: 144 (2006). Peptide deuterium levels weredetermined as described (Weis et al. J. Am Soc. Mass Spectrom. 17: 1700(2006)) using an HX-Express program. No adjustment was made fordeuterium back-exchange and results are reported as relative deuteriumlevel. Walsh, Posttranslational modification of proteins: Expandingnature's inventory, Ed. Roberts and Company Publishers, GreenwoodVillage, Colo. 1 (2006). Approximately 93% IgG1 amino acid sequenceidentity was confirmed by tandem MS. In total, the deuteriumincorporation of 222 peptic peptides was monitored. The error associatedwith each deuterium incorporation point was ±0.2 Da, anddifferences >0.5 Da were considered significant.

This technique monitors the exchange of amide hydrogens present in thepolypeptide backbone with hydrogen or deuterium in solution which isindicative of solvent exposure and hydrogen bonding, properties thatvary dramatically with protein structure. Coupled with enzymaticdigestion, the technique provides structural information localized tosmall regions of the protein (for example, short peptides spanning 3-10residues) and thereby reveals detailed information on proteinconformation in solution. Preparations of mAb-A containing low or highlevels of H-L trisulfide, or treated with 1 mM cysteine while bound toprotein A, were selected for analysis with the rationale that anyalterations in protein conformation would be revealed by differences inamide hydrogen exchange. When a total of 140 different peptic peptides,representing 94% sequence coverage, were analyzed within each IgG1 mAb-Apreparation, no differences in deuterium exchange were detected. Notablythis included the three peptic peptides encompassing the L and H chainCys residues that participate in the H-L and H-H inter-chain trisulfideor disulfide bonds. This indicated that high levels of H-L trisulfide,and on-column conversion of trisulfide to disulfide by cysteine, had nodetectable effect on mAb-A structure and folding.

Consistent with this, mAb-A preparations containing low or high levelsof H-L trisulfide, or treated with 1 mM cysteine, displayed similarfolding characteristics when analyzed by differential scanningcalorimetry. The DSC was performed using a MicroCal capillary VP-DSCsystem (Northampton, Mass., USA) with Origin VPViewer2000 v2.0.64software. Sample concentration was 0.5 mg/mL in 50 mM sodium phosphate,100 mM NaCl, pH 6.0. Thermograms were generated by scanning temperaturefrom 25° C. to 100° C. at a rate of 2° C./min. Data were processed usingOrigin 7SR2 software, and no differences in transition temperatureprofiles detected.

Example 8 Prevention of Trisulfides by Inhibition of CysteineDegradation

As shown above, hydrogen sulfide is present in the medium as a result ofcysteine degradation. It is hypothesized that the hydrogen sulfide whichforms from the feed medium is sufficient to induce trisulfide bondformation. To test this hypothesis, a small scale study was performed inflasks. Purified antibody with a known trisulfide content of 2% wasspiked into basal medium with and without the presence of cells. As anadditional comparison, cells were grown without the external addition ofantibody but with the addition of the antibody storage buffer. On thethird and fourth days of incubation, a 3% by volume addition of feedmedium was delivered. The antibody was harvested on the fifth day,followed by purification, concentration, and analysis by peptide mappingfor trisulfide content.

The amount of trisulfide bond formation is shown in in FIG. 7, comparingthe purified antibody used for spike experiments (1.8% trisulfidecontent) to the subsequently harvested material. Antibody produced bycells contained approximately 4% trisulfide content which is nearlyidentical to the trisulfide content when antibody was exogenously addedto the cells, despite the titer produced by cells representing onlyabout 5% of the total antibody at the end of the 5 day culture. Thecell-free condition contained spiked antibody with over 11% trisulfidecontent by day 5, which is over a six-fold increase from the startingcontent. It is clear from these data that incubation in medium withoutcells is sufficient to induce increases in trisulfide content, and thepresence of cells actually diminishes the effect of incubation inmedium.

Example 9 Reduction in Hydrogen Sulfide Release by Inhibitors ofCysteine Degradation

In order to test the effect of cysteine degradation inhibitors onrelease of hydrogen sulfide in cell-culture medium, various inhibitorswere added to the medium at the concentration of 17.7 mM, whichrepresents a 1:1 ratio to the concentration of cysteine in the medium.Released hydrogen sulfide concentration was measured after 24 hours ofincubation. Briefly, 50 ml of medium was incubated overnight in a closed100 ml bottle. The headspace was therefore equal to the liquid volume ofthe container. A Jerome 631-X hydrogen sulfide detector was connected toa short piece of silicon tubing to minimize holdup volume in theinstrument. The end of the tubing was inserted into the headspace of themedium-containing bottle, and the Jerome 631-X detector sampled theheadspace immediately after opening the bottle. FIG. 8 shows thatpyruvate, methyl pyruvate, ethyl pyruvate, DL-glyceraldehyde, andglyoxylic acid all functionally stopped release of hydrogen sulfide.

Example 10 Effect of Pyruvate on Release of Hydrogen Sulfide

The effect of pyruvate on release of hydrogen sulfide from cysteinedegradation was further tested. The experiments were performed asdescribed above in Example 9. Concentration of released hydrogen sulfidewas measured after incubation from solutions containing 17.7 mMcysteine, plus 200 g/L glucose or subgroups of amino acids, in theabsence or presence of of pyruvate. FIG. 9 shows that release ofhydrogen sulfide was significantly decreased when pyruvate was presentin solution.

Example 11 Effect of Pyruvate on Release of Hydrogen Sulfide in VariousMedia

The effect of pyruvate on release of hydrogen sulfide from cysteinedegradation was also tested in various cell-culture media, includingfeed liquid, IMDM and DMEM. All cysteine concentrations were normalizedto that in the feed liquid. 17.7 mM pyruvate was added to the medium,which represents a 1:1 ratio of cysteine to pyruvate in the medium. Theexperiments were performed as described above for Example 9. FIG. 10shows that pyruvate significantly reduced cysteine degradation (fromabout 60% to over 80% reduction) in all the media tested. Thus, pyruvatecan reduce cysteine degradation and H₂S release in a variety ofcompositions and decrease the number of trisulfide bonds.

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and any compositions or methodswhich are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

All documents, articles, publications, patents, and patent applicationsmentioned in this specification are herein incorporated by reference tothe same extent as if each individual publication or patent applicationwas specifically and individually indicated to be incorporated byreference.

What is claimed is:
 1. A method for reducing the formation of trisulfidebonds in proteins during large-scale production comprising culturingcells expressing said proteins in the presence of an effective amount ofan inhibitor of cysteine degradation, whereby trisulfide linkageformation in said proteins is reduced relative to cells cultured inmedium without the inhibitor of cysteine degradation, wherein theinhibitor of cysteine degradation is formaldehyde, acetaldehyde,propionaldehyde, butyraldehyde, stearic acid, arachidonic acid,docosahexaenoic acid, myristoleic acid, palmitoleic acid, elaidic acid,erucic acid, vaccenic acid, penicillamine, carotenes, alpha-tocopherol,ubiquinol, lactic acid, formic acid, oxalic acid, or uric acid, andwherein said cells are mammalian cells.
 2. The method of claim 1,wherein said mammalian cells are selected from the group consisting ofCHO (Chinese Hamster Ovary) (including CHO-K1, CHO DG44, and CHODUXB11), VERO, HeLa, (human cervical carcinoma), CV1 (monkey kidneyline), (including COS and COS-7), BHK (baby hamster kidney), MDCK, C127,PC 12, HEK-293 (including HEK-293T and HEK-293E), PER C6, NSO, W138,R1610 (Chinese hamsterfibroblast) BALBC/3T3 (mouse fibroblast), HAK(hamster kidney line), SP2/0 (mouse myeloma), P3x63-Ag3.653 (mousemyeloma), BFA-IcIBPT(bovine endothelial cells), RAJI (human lymphocyte)and 293 (human kidney) cells.
 3. The method of claim 1, wherein theratio of the inhibitor to cysteine is about 5:1 to 1:10.
 4. The methodof claim 1, wherein said inhibitor is added at a concentration ofbetween about 50 μM and about 500 mM.
 5. The method of claim 4, whereinsaid inhibitor is added at a concentration of between about 100 μM andabout 100 mM.
 6. The method of claim 5, wherein said inhibitor is addedat a concentration of between about 1 mM and 100 mM.
 7. The method ofclaim 6, wherein said inhibitor is added at a concentration of betweenabout 5 mM and about 50 mM.
 8. The method of claim 1, wherein saidinhibitor is added at the beginning of the culture period.
 9. The methodof claim 1, wherein said inhibitor is added during a feed in a fed-batchculture.
 10. The method of claim 1, wherein said cells are cultured in abioreactor.
 11. The method of claim 1, wherein said cells are grown insuspension.
 12. The method of claim 1, wherein said protein is anantibody or Fc-fusion protein.
 13. The method of claim 1, wherein saidcells are in IMDM or DMEM.